Key words
Steroid hormones - glucocorticoids - androgens - adrenocortical carcinoma - cell culture
Introduction
Only few cell culture models of the adrenal cortex have been established to study
molecular pathways in adrenocortical physiology and steroidogenesis and also to
develop treatments of adrenocortical carcinoma (ACC).
ACC is a rare malignant disease [1]
[2] which harbors a high risk of
recurrence and metastatic spread even after complete resection [3]
[4]
[5]. The human NCI-H295 cell line is the
current “gold standard” for in vitro experiments in ACC
research [6]
[7]
[8]
[9]
[10]
[11]. This cell line has also been of
great value in understanding human adrenal physiology [12]
[13] and toxicology of endocrine active
environmental polluants [14]
[15]
[16]
[17]. Importantly rodent animal models do
not fully reflect human properties of the adrenal cortex [18]. Only recently, Hantel et al [19] and Kiseljak-Vassilliades et al. [20] established new cell lines that were
termed MUC-1 and CU-ACC1/CU-ACC2, respectively.
The initial NCI-H295 cell line has been described to secrete predominantly androgens
and androgen sulfates (dehydroepiandrosterone- epiandrosterone- and
androsterone-sulfate). It was reported that growth and steroid production was 4-fold
decreased in serum-free medium [21].
As derivatives of these NCI-H295 cells, several substrains have been created by using
alternative culture conditions. These were designated as NCI-H295
“R” and grow as a monolayer ([Fig. 1]). NCI-H295 “R”-S1 requires Nu-Serum type 1,
NCI-H295 “R”-S2 is cultivated in the presence of
Ultroser® and NCI-H295 “R”-S3 was established
with Cosmic Calf serum [22]. A similar
strategy was pursued to obtain a strain named NCI-H295 “A” by
removing non-attached cells during passaging. In 2008 the cell lines “HAC
13/15” were established as monoclonal sub-strains from NCI-H295R
cells [23].
Fig. 1 Published sub-strains derived from the original NCI-H295
cells. NCI-H295 S1–3 sub-strains were established by cultivating
NCI-H295 cells over different periods and using various cell culture
additives. Floating cells were removed periodically to select adherent
cells.
NCI-H295 cells that grow as monolayers exhibit different responses to hormonal
stimulation. The initially described NCI-H295 cells did not respond to
ACTH-stimulation which is shared by the NCI-H295A strain. HAC 13/15 in turn are
sensitive to stimulation with ACTH while conflicting data have been reported for
NCI-H295R cells [24]
[25]
[26]. On the other hand, preserved
response of NCI-H295R cells to forskolin and the absence of ACTH-responsiveness
indicates a defective receptor signaling. Ang II is able to stimulate HAC
13/15 and NCI-H295R cells but does not lead to aldosterone secretion in
NCI-H295A and NCI-H295 cells, respectively [27]
[28]
[29]
[30].
It is well known that selection induced by prolonged growing and culture conditions
can lead to genetic, transcriptional and translational changes in cell lines and
lead to the development of substrains in different laboratories. [31]. This fact is illustrated by data
published by Samandari et al. who compared NCI-H295R with NCI-H295A cells and found
expression of 3β-hydroxysteroid-dehydrogenase (HSD3B2), sulfonyltransferase
and cytochrome b5 to be expressed at higher levels in NCI-H295A cells whereas higher
17, 20-lyase activity was present in NCI-H295R cells [26]. The observation of lower mRNA
expression of 3β-hydroxysteroid dehydrogenase in NCI-H295R cell lines
underscored their observation of an androgen dominated pathway in steroid
metabolism.
In summary these data suggest relevant differences in gene expression and phenotype
of cell lines in this standard model for in vitro experiments. Published data
are limited as to which extent experimental conditions, cell strains and passaging
impact on steroid excretion of NCI-H295 cells. Coming from discrepancies between
results in our laboratory and the literature, we here used liquid chromatography
mass spectrometry to address these questions by measurement of relevant steroid
hormones and metabolites in NCI-H295 cell culture supernatants in various
experimental settings.
Materials and Methods
Chemicals
All buffers, chemicals, substances and solvents were purchased from Merck
(Darmstadt, Germany) unless otherwise stated. DMEM/F-12, HEPES
w/o phenol red, Gibco™ Insulin-Transferrin-Selenium and ITS
premix CorningTM were from Fisher Scientific (Schwerte, Germany),
Corning Nu-Serum™ Growth Medium Supplement and Ultroser G™ were
purchased from PALL™ (Hampshire, UK). Reagents, standards, controls and
analytical HPLC-columns for mass spectrometry were obtained from
Chromsystems™ Chemicals (Gräfelfing).
Cell strains
NCI-H295R cells were obtained from Cell Line Service (CLS, Eppelheim, Germany)
and are designated in this study as “0” (passage 21) which was
used as reference cell line. Consecutive passages “A” (CLS,
passage 37), “B” (CLS, passage 55), “C” (CLS,
passage 46) of these cells, and cells designated “D” (passage
16) received from ATCC (Wesel, Germany) and E (unknown passage, continuous use
outside of our lab) were used. All these cells were grown as monolayers in F12
HAM-medium supplemented with 1 mg/ml insulin,
0.55 mg/ml transferrin, 0.67 µg/ml
selenium, and 2.5% Corning Nu-Serum, which served as the reference
medium in a humid atmosphere at 37°C and 5% CO2.
NCI-H295S suspension cells from ATCC were grown in RPMI 1640 medium supplemented
with 10% FCS, 1% Transferrin, 4 µg/ml,
0.04% Insulin.
Cells were authenticated by comparison of short-tandem-repeat (STR) profile with
the online database of the German Collection of Microorganisms and Cell Cultures
GmbH (DSMZ,
https://www.dsmz.de/services/services-human-and-animal-cell-lines/authentication-of-human-cell-lines.html).
Cells were cultivated for four days, supernatants were analyzed from parallel
cultures every 24 h each for steroids by LC-MS/MS.
Culture media
NCI-H295R cell line (cell line “0”) was grown in the medium
described above (reference medium) in a humid atmosphere at 37°C and
5% CO2.
To analyze the impact of culture media on steroidogenesis, cells were cultivated
in medium 0 (DMEM/F12supplemented with 2.5% Nu-Serum, 1%
GibcoTM ITS ), medium 1 (DMEM/F12 supplemented with
2.5% Nu-Serum, 1% GibcoTM ITS , 0.12% BSA),
medium 2 (DMEM/F12 supplemented with 2.5% Nu-Serum and
1% ITS Premix CorningTM), medium 3 (DMEM/F12
supplemented with 2% Ultroser G and 1% GibcoTM ITS)
and medium 4 (DMEM/F12 supplemented with 10% FCS and 1%
GibcoTM ITS) for four days and supernatant removed and analyzed
by LC-MS/MS as described above. On day 0, 106 cells from one
flask were distributed into two different well plates as biological duplicates.
Every 24 h, 500 µl from a total volume of 1 ml medium
was removed for analysis and an equal amount of fresh medium added. Every day
cells were counted and the absolute amount of steroids normalized to cell count.
(Countess II FL Automated Cell Thermo Fisher Scientific, Schwerte, Germany)
(Table 1S–5S).
Drug treatment
Consecutive passages 2 (ATCC2) and 4 (ATCC3) of cell lines
NCI-H295R (initial passage 9, ATCC1) from ATCC and consecutive
passages 12 (CLS1),14 (CLS2), 17 (CLS3), 74
(CLS4), 77 (CLS5) and 80 (CLS6) of cell
line NCI-H295R (initial passage 21) from CLS and consecutive passages of
NCI-H295S suspension cells (CLS7, CLS8, CLS9)
were treated with abiraterone (1 µM), metyrapone
(0.5 µM) or mitotane (10 µM) after cultivation
[9] for 48 h in
biological triplicates.
Steroid hormone profiling
For quantification of the 13 steroid hormones ([Fig. 2]) aldosterone, cortisone
corticosterone, progesterone, cortisol, 11-deoxycortisol,
17α-OH-progesterone (17α -OHP), dehydroepiandrosterone -sulfate
(DHEAS), testosterone, androstenedione, dehydroandrosterone (DHEA),
dihydrotestosterone (DHT) and estradiol, we used the “Mass Chrom
steroids®” assay (Chromsystems,
Gräfelfing) which was validated for cell culture supernatants (see
supplementary methods). Samples were treated according to the
manufacturer’s instructions. Briefly, 500 µl cell
culture supernatant were subjected to off-line solid -phase extraction in 96
well plates. After a cleaning and concentration step 15 µl of
each sample were injected into an Agilent 1290 U-HPLC coupled to a QTRAP
6500+(Sciex, Toronto) tandem mass spectrometer. Measurements were
performed in ESI+mode except for aldosterone and DHEAS (ESI-). Stable
isotope standards were used for all analytes and two MRM transitions
(qualifier/quantifier) were established. Quantification was performed by
6-point calibration curve (1/x weighting). Correctness of measurements
was confirmed by three quality control level running before and after each batch
and periodic participating in external ring trials and inter-laboratory
comparisons. Data were processed with Analyst® 1.6.3
Software. Results of the validation process in cell culture medium are supplied
in Supplementary Methods.
Fig. 2 Metabolism and synthesis of steroid hormones in adrenal
cells. Color coding is yellow for preliminary steps in glucocorticoid
synthesis, green for glucocorticoids and precursors, blue for
mineralocorticoids, orange for androgens and brown for estrogens.
Asterisks indicate steroid hormones quantified by LC-MS/MS in this study
(after Krone et al.[38])
Statistical analysis
All graphs, heat maps, figures and statistical analysis were created with Graph
Pad Prism® 7.00, 2016 (Graph Pad Software Inc., San Diego,
CA, USA). Mean±SD are provided. For comparison of means one-way ANOVA
with Sidak’s multiple comparisons Test was used. p<0.05 was
considered to be statistically significant.
Results
Impact of cell culture media on steroidogenesis
To determine the impact of cell culture media on steroidogenesis of NCI-H295R
cells, we used the cell line 0 and media 1–4, which had all been
previously described. In culture medium 2 ([Fig. 3]), we detected
0.19±0.001 µg/106 cells estradiol
on day 1, which increased during cultivation. Medium 3 contained approximately
8.1 µg/l estradiol, while in other media estradiol
content was lower than 93 ng/l. Steroid and hormone level of
cell free media can be found in supplemental Table 6S. When looking at
mineralocorticoid secretion, aldosterone differed between media up to ten fold
(medium 1 vs. medium 4) on day 3 (0.06±0.01
vs.0.55±0.02 µg/106 cells,
p<0.05). DHEA secretion differed by a factor of three between medium 3
and 4 on day 2 (4.89±0.68 vs.
13.45±0.66 µg/106cells,
p<0.05). Progesterone clearly decreased by 75% from day 2 to day
4 in medium 4 while 17α -OHP, 11-deoxycortisol, cortisol, cortisone and
testosterone increased, reflecting a predominant steroid metabolism pathway by
CYP21A2, 17A1 and 11B1. Consistently, corticosterone decreased clearly from day
2 to day 4 just as aldosterone by 30%. Overall, medium 1 led to a
decrease of the relative amount of glucocorticoids while, in medium 4 all
steroids except cortisone and estradiol were present at higher concentration in
relation to reference medium.
Fig. 3 Steroid patterns in NCI-H295R cells illustrate
heterogeneity in hormone excretion depending on composition of cell
culture media. Heat map analysis of LC-MS/MS data. The columns represent
time and the rows different media (1, 2, 3,
4) in relation to standard medium used. Values are mean of
biological duplicates. FC (fold change)
Impact of cell line supplier and passage on steroid secretion
By using culture medium 0 as a reference, we next investigated the impact of
passaging and cell line provider in terms of secreted steroid metabolome ([Fig. 4]).
Fig. 4 Steroid hormone profiles in cell culture supernatants from
NCI-H295R cells purchased from the CLS (cell lines A, B,
C, E) or ATCC (D) at various passages. CLS cell
line at passage 21 (designated as “0”, not shown) at
corresponding time points served as reference. The columns represent
cultivation time and the rows the analyzed steroids by liquid
chromatography mass spectrometry. Log10 fold changes relative
to cell number of NCI-H295R cells (CLS, substrain S1, passage 21) are
displayed with blue indicating decrease, red indicating increase.
A (NCI-H295R cells CLS passage 37), B (NCI-H295R cells
CLS passage 55), C (NCI-H295R cells CLS passage 46), D
(NCI-H295R cells ATCC passage 16), E (NCI-H295R cells CLS
external laboratory). Values are the mean of biological duplicates. FC
(fold change)
Between two passages of a single NCI-H295R cell line (Cell Line Service) in our
laboratory (A vs. B) aldosterone was found to be
0.316±0.02 µg/106 and
0.158±0.006 µg/106 cells (p<0.05)
on day 3, 11-deoxycortisol differed twofold (131.3±10.5 vs.
59.2±2.25 µg/106 cells, p<0.05)
and 17α -OHP almost two fold (10.5±0.51 vs.
6.3±0.16 µg/106 cells, p<0.05),
respectively. By comparison, cells purchased from ATCC (D) showed predominant
androgen secretion compared to CLS cells (A/B/C) as reflected by twice as high
secretion of DHEA (6.9±5 vs.
3.3±0.1 µg/106 cells, p<0.05),
three times more testosterone (15.9±0.51 vs.
5.2±0.29 ng/106 cells, p<0.05) and more
than double dihydrotestosterone (0.68±0.03 vs.
0.28±0.02 µg/106 cells,
p<0.05).
Throughout the whole test period, CLS cells (e. g. B) secreted up to
three-fold more estradiol compared to ATCC (D) cells (148±2.63 vs.
60±1.68 ng/106 cells on day one, p<0.05)
A significant increase over time in estradiol concentration was found in
supernatants from cell culture A, B and C (all CLS) in comparison to cell line E
with constant low secretion. A comparable trend was found for testosterone where
cell line D (ATCC) showed three times higher concentration on day 4 compared to
day 1(5.3±0.08 vs. 17.7±0.71 ng/106 cells,
p<0.05).
Cell line E produced a remarkably lower amount of steroids compared to all other
cell lines and passages but with a preponderance of estradiol and 17α -
OHP ([Fig. 4]).
To show the differences in relative amount and time course of steroid hormone
secretion more clearly, we here consider cell lines A and D in more detail
([Fig. 5]). Whereas in cell line
D secretion of most hormones increased continuously over cultivation time, the
concentration of androstenedione reached a plateau after one day of growing in
both cell lines (55.6±025 vs.
87.4±0.06 ng/106 cells, p<0.05).
The absolute quantities of steroid hormones differed markedly. Thus, on day 3
cell line D reached a maximum 17 α -OHP concentration of
26.8±0.79 ng/106 cells while less than
half (10.5±0.51 ng/106 cells) was found in
CLS cells on the same day. Progesterone was detected at higher concentrations
early during cultivation which then decreased over four days to less than
15% of baseline level.
Fig. 5 Steroid hormone excretion profile in NCI-H295 R cells from
different providers. Quantity of 13 steroid hormones (aldosterone,
corticosterone, progesterone, cortisol,
cortisone,11-deoxycortisol,17α-OH-progesterone (17α
-OHP), dehydroepiandrosterone -sulfate (DHEAs), testosterone,
androstenedione, dehydroepiandrosterone (DHEA), dihydrotestosterone and
estradiol) was determined in cell culture supernatants by LC-MS/MS
normalized to cell count. Values are the mean of biological duplicates
which are indicated individually.
Over time, amounts of progesterone, corticosterone and aldosterone decreased in
cell line A whereas conversely we observed an increase of dihydrotestosterone
(0.142±0.004 µg/106 on day 1 vs.
0.265±0.028 µg/106 cells on day 4, n.
s.), testosterone (0.024±0.0003 µg/106 on day
1 vs. 0.053±0.0069 µg/106 cells on day 4,
p<0.05), cortisol (45.1±0.07 µg/106
on day 1 vs. 121.8±10.7 µg/106 cells on day
4, p<0.05), and cortisone
(1.16±0.001 µg/106 on day 1 vs
3.80±0.44 µg/106 cells on day 4,
p<0.05) as a marker of predominant metabolism of precursors by CYP17A1
and CYP11B1 to androgens and glucocorticoids under the selected conditions.
Differences in the ratios of cortisol and aldosterone between cell lines
illustrate heterogeneity in preference of 17α-hydroxylase over
3β-hydroxysteroid-dehydrogenase ([Fig. 6]). Cell line E clearly shows the highest
17α-hydroxylase conversion (ratio 1722±73) while the other CLS
cells in general prefer the mineralocorticoid way via HSD3B2. The strong
17α-hydroxylase activity of cell line E is underlined by comparing
17α-OHP/progesterone ratios which is lowest (ratio
108±2) in cell line C. The most significant relative conversion activity
via 17,20 lyase can be found in cell line B. By comparing diagnostic ratios of
17α-hydroxylase and17,20 lyase reaction rates, cell line A dominates
represented by ratio of 0.038±0.0005.
Fig. 6 Diagnostic ratios of steroid concentrations in different
cell lines (NCI-H295R cells purchased from CLS (cell lines A,
B, C, E) or ATCC (D) reflecting enzyme
activities of 3β-hydroxysteroid-dehydrogenase (HSD3B2) and
17α-hydroxylase and 17, 20-lyase. Values are mean of biological
duplicates.
Manipulation of steroidogenesis with inhibitors
Three consecutive early and late passages of NCI-H295R cells from cell line 0,
three passages of ATCC cells and NCI-H295 suspension cells were treated with
inhibitors of steroidogenesis ([Fig.
7]). The CYP11B1 inhibitor metyrapone led to a decrease of
aldosterone, cortisol, cortisone and corticosterone, which was however not
apparent in some adherent cells (CLS4, CLS5,
CLS6) and suspension cells (CLS7, CLS8,
CLS9) where cortisol and corticosterone remained almost
unchanged. increase in 11-deoxycortisol, testosterone and dihydrotestosterone
concentration was only observed in CLS4, CLS5 and
CLS6 adherent cells and in suspension cells indicating an impact
of passaging on steroidogenic properties.
Fig. 7 Steroid pattern of various NCI-H295R (S) cell lines
illustrate the heterogeneity in response to CYP 11B1 (metyrapone) and
CYP17A1 (abiraterone) inhibition and ER-stress, respectively induced by
mitotane. Heat map analysis of LC-MS/MS data. The columns represent the
measured steroids normalized on cell count and the rows the various
NCI-H 295 cell lines and passages. Cells were exposed to metyrapone
(0.5 µM), abiraterone (1 µM) and
mitotane (10 µM) for 48 h. Values are mean of biological
triplicates. FC (fold change)
Inhibition of CYP17A1 by abiraterone showed a consistent increase of aldosterone,
corticosterone and progesterone while glucocorticoids and androgens decreased in
all cell lines. In suspension cells aldosterone, corticosterone and progesterone
production was almost unchanged. A massive decrease of all steroids was induced
by mitotane reflecting the interference in early state of cholesterol metabolism
and endoplasmic reticulum stress. In three passages of CLS (CLS4,
CLS5 and CLS6) cells we found an increasing amount of
testosterone compared to unexposed cells.
Discussion
Our experiments clearly show that secretion of steroids is influenced by the provider
of NCI-H295 cells, passaging and cell culture media. While prolonged passaging is
known to change properties of cell lines, phenotypic consequences of media are less
well understood.
Even though all cell lines investigated are based not only on the same original cells
but also derive from the same substrain ([Fig.
1]) NCI-H295R S1, the extent of changes in steroidogenesis was
striking.
In early experiments Gazdar et al. in 1990 already analyzed steroid secretion of
NCI-H295 cells by GC-MS and photometric methods [21] and found higher secretion of cortisol
and androstenedione in medium containing 2% bovine serum which was similar
with regard to most other steroids and consistent with an overall stimulation of
steroidogenesis. Although the exact amount of steroids normalized to cell count is
not exactly comparable, they found 0.5 µg/106
cells cortisol, 0.4 µg/106 cells androstenedione
and 0.1 µg/106 cells DHEA which is less than
10% of the quantity in our experiments. Despite heterogeneity, overall the
complete spectrum of adrenal androgen and glucocorticoid pathways was expressed
(CYP11A1, CYP17A1, CYP21A2, CYP11B2, CYP11B1) [22] which is in accordance with the results by Gazdar et al. [21] that were obtained 10 years after
establishing the initial cell line.
Wang et al. compared aldosterone production among different human adrenocortical cell
lines including S1–3 substrains of NCI-H295R cells [32] at the level of steroidogenesis and
mRNA expression of key enzymes and showed CYP11A1, HSD3B2, CYP11B1 and CYP11B2
expression with remarkable difference between the S2 and S3 substrains of NCI-H295R
cells. These cell strains differ only in the adaption to cell culture medium
composition ([Fig. 1]).
In our hands, ATCC passage 16 cells exhibited a clear predominance of progesterone,
17α -OHP, androstenedione, DHEA, testosterone and DHT but also
glucocorticoids in comparison to reference cell line (CLS, substrain 1, passage 21).
This observation is consistent with data presented by Hornsby et al. [33] who showed a decline in CYP17A1
activity related to senescence. Hence it appears that the relative increase of
aldosterone and corticosterone in CLS cells might reflect reduced
17α-hydroxylase/lyase activity but also may impair overall steroid
production. Ratios of marker substances representing hydroxylase and lyase
activities suggest a predominant pathway to androstenedione via
17-hydroxypregnenolone and DHEA. However, as pregnenolone and 17-hydroxypregnenolone
were not included in our measurements, the approach provides only partial insight
into the entire steroidogenic network.
We cannot explain all aspects of steroid hormone secretion without detailed enzyme
expression data available. For example, relevant amounts of cortisone suggest the
in vitro activity of 11β-hydroxysteroid dehydrogenase and
5-α reductase appears to be active in some strains (A/D) as
indicated [24] by considerable quantities
of DHT. With the exception of DHEAS, our assay does not capture steroid hormone
metabolites. Presence of sulfotransferases may explain the decline of some steroids
such as estradiol over time in a subset of our experiments. Care should be taken in
choosing cells reflecting best the targeted approach and research question.
Exposure of NCI H295 cells from different origins to abiraterone, metyrapone and
mitotane was consistent with the overall expected patterns that are explained by the
respective mechanism of each drug. However, in early passage cell lines treatment
with metyrapone e. g. did not lead to increased 11-deoxycortisol and
androgen -secretion, which was the case in later passage cultures.
There are several possible explanations such as less active steroidogenesis in
earlier passages leading to less pronounced accumulation of precursors and diversion
to androgens or off target effects of inhibitors [34]. Since in the in vitro system
used, feedback through corticotropin is absent and cAMP/PKA was not
experimentally stimulated, increase in 11-deoxycortisol observed upon metyrapone
incubation in some cell lines may indicate autonomous PKA activation in NCI-H295
cells.The decrease in glucocorticoids and androgens and switch to mineralocorticoids
in the presence of abiraterone suggests relevant inhibition of 17-alpha-hydroxylase
(and not only 17,20-lyase). Consequences of abiraterone treatment in vivo are
well known and include a rise of mineralocorticoids. However these in vivo
results are not transferable to our experiments as stimulation through feedback
mechanisms is absent [35] as described
above. Recently, canine adrenal cells were tested as a model to study the impact of
abiraterone on steroidogenesis [36]. In
accordance with our results abiraterone treatment led to elevation of progesterone
and decrease of 17α -OHP, 11-deoxycortisol, cortisol, androstenedione and
cortisone but in that system, also to reduced aldosterone and corticosterone
production ([Fig. 7]).
Finally mitotane blocked steroidogenesis pathway in NCI-H295 cells at an early stage
which resulted in decreased levels for nearly all steroids consistent with the
recently described down-regulation of steroidogenesis by accumulation of free
cholesterol through SOAT1 inhibition and subsequent blockage of sterol responsive
genes. This appears to depend on both down-regulation of sterol-responsive element
binding transcription factors and induction of endoplasmic reticulum stress [9]. Our results do not support a direct
influence on steroidogenic enzymes similar to recent reports [37].
For decades, NCI-H295 cells have been the only model for human adrenal physiology and
the development of sub-strains enabled clarification of aspects related to
mineralocorticoid, glucocorticoid and androgen synthesis. However the unavailability
of alternative human cell line models has been a serious obstacle in ACC research
since results could not be reproduced in other cellular contexts.
Observed heterogeneity may be partly explained by evolutionary effects occurring in
cultivated cell lines. Accordingly, Uri Ben-David et al. described extensive
variation in response of 27 MCF7 cell strains to 321 different drugs which was
related to gene expression and transcriptional pattern [31].
The landscape of human ACC cell line models is starting to change with the
description of MUC-1 cells [19] and
CU-ACC1 and 2 cells [20]. These reflect a
broader range of the biological spectrum of ACC and will enable independent
verification experiments. It is necessary to mention, however, the artificial
upregulation of steroidogenesis e. g. by addition of cholera toxin to the
culture medium with the aim to increase cellular cAMP content. This may limit the
utility of some cell lines for the study of adrenal physiology.
Moreover our study explains the partly divergent results obtained in the presumably
identical cell line and uses steroids as surrogate markers of signaling cascades. It
is generally acknowledged that prolonged passaging changes properties of cell lines
but consequences of culture in different media are less well understood. Therefore
our findings provide additional evidence that care must be applied when choosing
culture conditions in particular since effects are often more difficult to assess
compared to steroidogenesis. LC-MS/MS is an ideal tool to comprehensively
characterize steroidogenesis and to ensure reproducibility. Our findings can be
summarized in practical recommendations ([Table 1]) for cell culture experiments focusing on steroidogenesis.
Table 1 practical recommendations for experiments using NCI-H295
cell lines focusing on steroidogenesis
|
factor influencing sterodogenesis
|
consequence
|
|
cell (sub)strain
|
careful selection of cell (substrain) use of the same (sub)strain
whenever [possible
|
|
passage
|
use of same/similar passage periodic measeurement of
secretory activity.
|
|
vendor
|
use the same source/vendor
|
|
medium
|
choose of medium depending on steroidognic pathway under study
assessment of staroidogensis preferably by LC-MS/MS when
supplier/batch changes
|
|
steroid hormone content of media
|
use of the same medium whenever possible blank medium required
for experiments assessing steroid synthesis, normalization of
results on initial quantity of hormones/steroids
|
