Characterization of NCI-H295R Cells as an In Vitro Model of Hyperaldosteronism
Authors
U. D. Lichtenauer1, I. Shapiro1, A. Osswald1, S. Meurer2, A. Kulle3, M. Reincke1, F. Riepe3, F. Beuschlein1
Affiliations
1 Endocrine Research Unit, Medizinische Klinik und Poliklinik IV, Klinikum der LMU, Munich, Germany
2 Endocrine Laboratory, Medizinische Klinik und Poliklinik IV, Klinikum der LMU, Munich, Germany
3 Pediatric Clinic, University Hospital Kiel, Kiel, Germany
Key words
· aldosterone producing cell line Conn’s syndrome adrenal tumor
Abstract
▼
In depth analysis of key molecular mechanisms involved in functional autonomy of aldosterone secretion is hampered by the lack of tumor cell lines that reflect functional characteristics of aldosterone producing adenomas. Herein, we describe the characteristics of the adrenocortical carcinoma cell line NCI-H295R and its suitabil- ity as a model of hyperaldosteronism in relation to different culture conditions. Steroid profiling revealed that NCI-H295R cells predominantly secrete cortisol, while aldosterone and other steroids are released at much lower concentra- tions. However, aldosterone output specifically increased in response to different stimuli such as ACTH and angiotensin II, and in particular to potassium in a dose dependent manner. NCI-
H295R cells readily formed spheroids under spe- cific culture conditions, a method widely used for the enrichment of progenitor cells. Unex- pectedly, spheroid cells excelled with higher aldosterone concentration and higher expres- sion levels of the steroidogenic enzymes StAR, 3฿HSD, CYP17, SF-1, and the MC2-receptor. Fur- ther investigations revealed that this phenom- enon is mainly attributed to epithelial growth factor (EGF) and particularly fibroblast growth factor (FGF), which are both essential ingredients in the spheroid culture medium. Aldosterone release under the combinatory influence of EGF and FGF was not higher than the effect of FGF alone. Spheroid growth per se, therefore, does not ensure an enrichment of less differentiated cell types in this cell line.
received 10.06.2012 accepted 20.08.2012
Bibliography
DOI http://dx.doi.org/ 10.1055/s-0032-1323810 Published online: October 30, 2012 Horm Metab Res 2013; 45: 124-129 @ Georg Thieme Verlag KG Stuttgart . New York ISSN 0018-5043
Correspondence F. Beuschlein, MD Endocrine Research Unit Medizinische Klinik und Poliklinik IV Klinikum der Ludwig- Maximilian University Ziemssenstraße 1 80336 Munich Germany Tel .: +49/89/5160 2110 (2116) Fax: +49/89/5160 4467
felix.beuschlein@med. uni-muenchen.de
Introduction
▼
Primary aldosteronism (PA) has been recognized as the most common cause of secondary hyper- tension, which is responsible for elevated blood pressure in 5-10% of all hypertensive patients [1]. While the classical hypokalemic form of PA is prevalent in less than 0.5% of patients with hypertension, more sensitive diagnostic screen- ing approaches allow for the detection of the normokalemic variant of the disease which resulted in an increase of case detection and, thus, identification of PA as a relatively common disease [2].
On the contrary, the mechanisms accounting for autonomous aldosterone secretion are still only partly understood. All models currently being used in preclinical research have limitations. In vitro, the human NCI-H295R cell line is most commonly utilized [3,4], although it has been derived from an adrenocortical carcinoma and therefore inherently might not reflect the molec-
ular and functional changes of true aldosterone producing adenomas [5]. Nevertheless, in con- trast to other adrenocortical cell lines, such as the SW13 cells, these cells are known to be capable of aldosterone production to some extent. The aim of this study was to increase the knowledge about the characteristics of aldosterone produc- tion in NCI-H295R cells and their steroid profile under different culturing conditions.
In previous studies, the existence of subpopula- tions with distinct characteristics within a given tumor has been described. Some of these sub- populations seem to represent cells with progen- itor or stem cell-like properties, and were claimed to be responsible for most of the tumor growth [6,7]. Interestingly, these subpopulations could also be found in clonally expanding cell popula- tions, such as cell lines [8,9]. Therefore, besides analyzing the feasibility of NCI-H295R cells as an in vitro model for aldosterone excess, we investi- gated, whether a subpopulation of cells within the NCI-H295R adrenocortical carcinoma cell
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line could be enriched by sphere formation, a process originally described to enrich for neural and other progenitor cells, includ- ing the adrenal medulla, and stem cell-like cancer cells [6,8, 10- 13].
Materials and Methods
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Cell culture conditions
NCI-H295R monolayer cells and SW13 cells were grown at 37 ℃ and 5% CO2 in DMEM/F12 medium (Gibco Invitrogen, Carlsbad, CA, USA) containing 10% FBS (HyClone, South Logan, UT, USA) and 2 mM L-glutamine (BioWhittaker Cambrex, East Rutherford, NJ, USA), and 10ul/ml Pen/Strep (Gibco) as described previously [14]. Spheroid growth was induced by culturing the cells with- out serum in DMEM/F12 medium supplemented with B27, N2, 20 ng/ml hEGF, 20 ng/ml bFGF, and 10 ul/ml Pen/Strep (all Gibco). To measure growth factor effects on aldosterone release, either EGF or FGF was withheld from the medium in comparison to medium containing both factors, and in comparison to regular cell culture medium. For stimulation experiments, NCI-H295R monolayer cells were plated at a density of 50 000 cells in 24-well plates. After 72h, the medium was replaced, and ACTH, angi- otensin II, and potassium chloride (all Invitrogen) were added to 3 wells per stimulant at various concentrations (ACTH: 20nM, angiotensin II: 20 nM, potassium chloride: 5.2, 7, 9, 12, 15, 20, and 40 mM total concentration).
Steroid profiling
Mineralocorticoids, glucocorticoids, and androgens in the super- natant of NCI-H295R cells grown for 24h under standard cell cul- ture conditions and after stimulation with 20mM potassium, were measured in triplicates by ultra-pressure liquid chromato- graphy tandem mass spectrometry (UPLC-MS/MS) according to a previously described method [15]. In brief, aliquots of samples, calibrators, and controls were extracted by solid-phase extrac- tion (SPE) using Oasis MAX SPE system plates (Waters, Milford, MA, USA). Deuterium labeled steroids were used as internal standards. The UPLC-MS/MS system was used in the multiple reaction monitoring mode (MRM), and steroids were measured in the positive ion mode except aldosterone, which was meas- ured in the negative mode. For each hormone 2 different MRM transitions were monitored. The limit of quantification was between 0.1 nmol/l for 17-hydroxyprogesterone and 2 nmol/l for cortisol. The intra- and interassay coefficient of variations for replicate quality controls for different concentrations range between 2.4% and 9.7%. Total run time for the assay was 5 min.
Aldosterone assay
Aldosterone in cell culture supernatants was measured by an inhouse immunofluorometric assay, which initially was deve- loped for the measurement of aldosterone in saliva [16]. Samples were run in triplicates, and for comparative aldosterone release studies between spheroid and monolayer cells, results were nor- malized to total cellular protein. To exclude matrix effects, cell culture supernatants were extracted with dichloromethane and reconstituted in an artificial saliva matrix prepared according to the German Industry Norm (DIN 53 160) standards (4.2 g/l NaHCO3, 0.5 g/l NaCl, 0.2g/l K2CO3). The same matrix was also used to produce standards and to dilute samples with very high aldosterone concentrations.
Tissue staining and immunohistochemistry
NCI-H295R cells were grown on Coverslips (Becton Dickinson, Franklin Lakes, NJ, USA) for 5 days and fixed with PFA for at least 1 h. Spheroid cells were collected with a 100um mesh, fixed with PFA, dehydrated, and paraffin embedded. Tissue slides and coverslips were subsequently investigated by H & E staining. For immunohistochemical analysis, cover slips and tissue sections were rehydrated, blocked with 0.3% H2O2 in methanol for 10 min, and incubated with blocking buffer for 15 min. P450SCC primary rabbit anti-human antibody (kindly provided by Dr. Walter Miller, UCSF, CA, USA) was incubated overnight at 4℃ after dilu- tion in blocking buffer containing 3% bovine serum albumin (Sigma, St. Louis, MO, USA), 5% goat serum (Jackson Immuno Research Laboratories, West Grove, PA, USA), and 0.5% Tween 20 (Calbiochem, San Diego, CA, USA). After rinsing for 15 min in PBS, secondary antibody (Goat anti-rabbit biotinylated IgG; Vector Laboratories, Burlingame, CA, USA) was applied for 30min at room temperature. Bound primary antibody was detected using the VECTASTAINE Elite ABC Kit (Vector Laboratories) according to the manufacturer’s protocol.
Real-time PCR
RNA from cell preparations was extracted using the Qiagen RNA mini kit (Qiagen, Valencia, CA, USA) following the instructions of the manufacturer. cDNA was transcribed using a reverse tran- scription kit (Promega, Mannheim, Germany) and 1.0µg of total RNA.
Real-time PCR was performed using the FastStart DNA Master- Plus SYBR Green I reaction mix in the LightCycler 1.5 (Roche, Mannheim, Germany). The cycling conditions for real-time PCR included a preincubation step at 95℃ for 10 min, followed by an amplification step, which consisted of 40-45 cycles at 95℃ for 10s, annealing (the temperature was primer dependent as given below) for 6s and an extension step at 72℃, for which the time was calculated by the product length in bp divided by 25 (Roche, Indianapolis, IN, USA). Primer sequences as well as product lengths and annealing temperatures were as follows: P450SCC: 5’-GCAACGTGGAGTCGGTTTAT-3’ and 5’-TCCTCGAAGGACATCTT- GCT-3’ (664bp; 52℃); StAR: 5’-CAGGACAATGGGGACAAAGT-3’ and 5’-ATGAGCGTGTGTACCAGTGC-3’ (608 bp; 63℃); SF-1: 5’-TGCACTGCAGCTGGACCGCCAGGAGTT-3’ and 5’-AGGGCTCCT- GGATCCCTAATGCAAGGA-3’ (390bp; 58℃); 3ßHSD 5’-CTAAGT- TACGCCCTCTTCTG-3’ and 5’-AATGTCTCCTTCAAGTACAGT-3’ (285 bp; 50℃); Cyp 17 5’-CAA GCC AAG ATG AAT GCA GA-3’ and 5’- CAT AAA CCG ATC TGG CTG GT-3’ (438bp; 50℃); MC2- receptor 5’-CATGGGCTATCTCAAGCCAC-3’ and 5’- GAGATCT- TCCTGGTGTGGGATC-3’ (360bp; 55℃); CYP11B2 synthase 5’-GGGAGCGTGGACACGACAGC-3’ and 5’-GGCATTGCGACCCAGC- GAGT-3’ (303 bp; 60℃). Melting curve analysis was performed between 65 and 98 ℃ (0.1 ℃/s) to determine the Tm of the ampli- fied product and in order to exclude undesired primer dimers. Furthermore, the products were run on a 1% agarose gel to verify the size of the amplified product. Each sample was run at least in triplicate. Quantification was normalized using HPRT (hypoxan- thine phosphoribosyltransferase) as a housekeeping/reference gene. cDNA of 8 normal adrenals were pooled in equal volumes to achieve a representative standard for the expression analysis part of this study.
Statistical analysis
All results are expressed as mean + SD. If not stated otherwise, all statistical comparisons were analyzed by one-way ANOVA and
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independent samples T test. Statistical significance was defined as p<0.05 and is indicated as a star (*) in the figures.
Results
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Steroid profile
At baseline, ultra-pressure liquid chromatography tandem mass spectrometry revealed that cortisol was the predominantly secreted hormone in the supernatant of NCI-H295R cells (58.0±5.1 ng/dl), whereas the other steroid hormones were released on a much lower level in similar range (· Fig. 1a). Inter- estingly, potassium stimulation resulted in a distinct shift in the steroid profile due to a selective and significant 19.2-fold increase of aldosterone secretion ( Fig. 1b) while most other steroid metabolites were not significantly changed in a potas- sium dependent manner.
Aldosterone output of NCI-H295R cells at baseline and upon stimulation
Baseline aldosterone levels measured in the supernatant of NCI- H295R cells grown as monolayers after 24h of incubation ranged between 328-716 pg/ml, and were found to be responsive to
stimulation by angiotensin II (131±1.2% vs. 100±6.2%; p<0.01), while incubation with ACTH increased aldosterone secretion only slightly (118±6.4%; p=0.17; data not shown). However, the most pronounced aldosterone induction was observable upon potassium stimulation when aldosterone output increased from 352±8.64pg/ml at physiological potassium concentrations (5.2mM) up to 8407±1013 pg/ml at potassium concentrations of 20mM in a dose dependent manner (· Fig. 1c). Likewise, CYP11B2 expression gradually increased with increasing potas- sium concentrations up to a maximum of 411 ±0.3% at a potas- sium concentration of 15mM (· Fig. 1d). Further increase in potassium supplementation resulted in lower aldosterone con- centrations and lower CYP11B2 expression levels, respectively, most likely due to potassium dependent cell death ( Fig. 1c, d).
Spheroid versus monolayer growth
NCI-H295R cells were exposed to 2 different culture conditions: Regular serum containing medium and serum free medium sup- plemented with epithelial growth factor (EGF) and fibroblast growth factor (FGF). Adherent monolayer growth was observed (day 2 after plating), when cells were cultured in regular, serum containing medium, whereas they adopted spheroid-like struc- tures after plating a minimum of 100 cells per well on a 24-well
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a
regular medium
stem cell medium
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dissociation and 9 more days in culture
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plate in EGF and FGF containing serum free medium for a mini- mum of 3 days ( Fig. 2a, upper panels; · Fig. 2b, upper panels). NCI-H295R spheroids could be trypsinized and dissociated into single cell suspension and continued to grow as monolayer cul- ture, or re-formed spheroids at a much slower rate (23 more days vs. 9 days), depending on the culture conditions they were exposed to ( Fig. 2a, lower panel). Without dissociation, sphe- roid growth decreased over time and seized after approximately 2 weeks (data not shown).
Fixed and embedded spheroids and monolayer cells clearly stained positive for P450SCC in immunohistochemical analyses ( Fig. 2b, lower panels) indicating persistence of steroidogenic properties independent of culture conditions.
Steroidogenic capacity of spheroids
Baseline aldosterone levels secreted by spheroids (S) and nor- malized to total cellular protein were significantly higher in comparison to monolayer (M) cells (3409±644.9 vs. 615.7±11.9 pg/ml/mg/ml total protein; p=0.01; · Fig. 3a). Real-time PCR analyses of NCI-H295R cells grown as monolayers or spheroids revealed that expression levels of steroidogenic enzymes - nor- malized to HPRT - were approximately 100 times lower in NCI- H295R cells in comparison to pooled normal human adrenals (N): 3B-HSD, 0.36±0.02%; p<0.01 (S) and 0.01±0.0%; p <0.01 (M) vs. 100±19.5%(N),p450SCC:6.9±0.15%; p<0.01(S)and6.29±0.19%; p<0.01 (M) vs. 100±3.14% (N); StAR: 7.65±1.08%; p<0.01 (S) and 0.91±0.24%; p<0.01 (M) vs.100±9.29% (N); Sf-1: 22.65±1.11%; p<0.01(S)and 15.0±0.3%; p<0.01(M)vs.100±1.95%(N); Cyp17: 0.19±0.01%; p<0.01 (S) and 0.14±0.01%; p<0.01 (M) vs. 100±1.54% (N); MC2-R: 45.8±0.33%; p<0.01 (S) and 0.8±0.01%; p<0.01 (M) vs. 100±2.17% (N). Notably, with the exception of P450SCC, expression levels of all investigated genes were either equal or higher in spheroids in comparison to monolayer cells. In
contrast, no expression of any steroidogenic enzyme analyzed was detectable in SW13 cells, which is in agreement with their known lack of steroidogenic properties (· Fig. 3b).
To investigate the rationale behind the different steroidogenic properties of NCI-H295R cells grown as monolayer vs. spheroids, monolayer cells were incubated with the same concentrations of growth factors as used for spheroid growth induction. While the addition of EGF alone already led to a significant increase of aldosterone release (2529±255.4 vs. 1206+44.4; p=0.01), FGF treatment had an even greater effect, with aldosterone levels similar to those observed for spheroid cells (4779+435.4; p<0.01). The combination of EGF and FGF did not result in a response greater than FGF treatment alone (4219±22.1; p=0.35). Vice versa, when spheroid cells were switched to regu- lar monolayer culture medium without growth factors, aldos- terone concentrations in the supernatant dropped to a level similar to that of monolayer cells (1013±31.4; p=0.04; · Fig. 4). To exclude differences in aldosterone concentrations from dif- ferent amount of cells, results were related to total cellular pro- tein levels for all conditions mentioned.
Discussion
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Despite a number of potential drawbacks inherent to cell line dependent model systems, adrenocortical NCI-H295R cells are well established for their maintained steroid capabilities. Herein, we specifically analyzed the suitability of these cells as an in vitro model of autonomous aldosterone excess. Recently, endo- crine properties of different strains, or subtypes of NCI-H295R cells have been described [17]. These studies provided evidence that phenotypic variability and shifts through clonal selection have to be taken into account for the interpretation of experi- mental results. In addition, as shown herein, cell culture condi- tions that might induce stem cell like properties have to be considered as modifiers of secretory abilities.
At baseline conditions, cortisol was the most abundantly secreted hormone, which was expected and reflects the physio- logical ratio of steroid hormones from the adrenal cortex. Inter- estingly, potassium stimulation led to a relative decrease in cortisol output. Overall, potassium induced effects on adrenal glucocorticoid secretion are not well defined and clearly influ- enced by species-, cell- and context- (in vivo vs. in vitro) depend- ent mechanisms with examples of potassium induced increased [18], decreased [19], or unchanged [20] glucocorticoid levels.
While aldosterone output could be stimulated to some extent by ACTH and angiotensin II, incubation with high levels of potas- sium was followed by a pronounced, specific and exclusive shift towards mineralocorticoid production, as exemplified in a mul- tisteroid analysis. Accordingly, CYP11B2 expression as the rate limiting step of aldosterone production increased with rising potassium concentrations. However, the stimulatory response set in on supra-physiological potassium concentrations, a com- mon finding in cell line experiments. Concentrations higher than 15-20mM led to a decrease in CYP11B2 expression and aldosterone concentration, most likely due to hyperkalemia induced cell death, as the cells had a different appearance and partially detached from the bottom of the dish.
Since spheroid formation has been recognized as a tool to enrich neural precursors and stem cell like cancer cells, even in the con- text of mainly clonally expanding cells [8,9,12], we aimed to investigate, whether spheroid growth could be induced also
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Adrenal Mix
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from adrenocortical cells. In fact, in the presence of EGF and FGF in a serum-free medium NCI-H295R cells readily formed cell clusters, which progressively adopted the typical spheroid appearance. After dissociation and re-plating, the cells regrew as spheroids or as adherent cells, depending on the cell culture conditions they were exposed to. While we expected a less dif- ferentiated phenotype, surprisingly, spheroids seemed to retain the ability to produce steroids, as they expressed even higher levels of most of the steroidogenic enzymes tested, had greater aldosterone output, and grew slower than monolayer cells. Therefore, as confirmed by immunohistochemistry, RT-PCR and hormone measurements, spheroids from NCI-H295R cells seem to enrich for a rather differentiated phenotype.
In further experiments we analyzed potential direct effects of different culture conditions on spheroid and monolayer cell growth. Interestingly, when monolayer cells were exposed to the growth factors of spheroid medium, especially to FGF, their responsiveness to potassium significantly increased to a similar level as observed in spheroid cells. Vice versa, spheroid cells cul- tured in monolayer medium were characterized by aldosterone concentrations in the supernatant similar to that of monolayer cells. Taken together, most of the observed differences in aldos- terone release between monolayer and spheroid cells seem to be attributable to FGF in the spheroid cell culture medium. This
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further confirmed the results of the expression analyses, that spheroid growth per se, does not necessarily ensure an enrich- ment of progenitor or stem cell like tumor cells in all cell types.
In conclusion, we have demonstrated the ability of NCI-H295R cells to specifically respond to different stimuli of the mineralo- corticoid regulatory system, especially potassium, with a spe- cific increase in aldosterone output. In addition, the cells easily grew spheroids, however, adopted surprisingly a rather differen- tiated phenotype, which is most likely related to FGF exposure. The highest aldosterone concentrations achieved were still approximately 50 times lower than those measured within the first 24h of primary cells from aldosterone producing adenomas (data not shown). This finding underlines the need to continue the search for alternative, potentially more potent cell lines, preferentially from an aldosterone producing adenoma.
Conflict of Interest
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The authors have nothing to disclose and have no conflict of interest.
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