Prostate Cancer and Prostatic Diseases (1999) 2, 257±263
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Paper
Isolation and partial
characterization of an epithelial
cell line (RPE-F344) from the
regenerating prostate of a normal
adult male rat
SC Presnell1, WJ Glover1, KM Borchert1, CW Gregory2, JL Mohler1,3,4 & GJ Smith1,3*
1
Department of Pathology and Laboratory Medicine, UNC-Chapel Hill; 2Department of
Pediatrics, Laboratories for Reproductive Biology, UNC-Chapel Hill; 3UNC-Lineberger
Comprehensive Cancer Center; and 4Department of Surgery, Division of Urology, UNCChapel Hill
Normal prostate epithelial cells are dif®cult to propagate in vitro without
experimental immortalization. The goal of this study was to isolate and characterize a propagable epithelial cell line from normal adult rat prostate. Enrichment of proliferation-competent cells was accomplished in vivo by initiating a
single cycle of prostatic involution/regeneration. The RPE-F344 cell line was
established from an androgen-deprived, involuted prostate four days after the
initiation of regeneration by administration of testosterone. The cell line has
been cultured in vitro for > 50 passages, forms a uniform monolayer in culture,
exhibits contact inhibition at con¯uence, and does not form colonies in soft agar.
Immunocytochemical and RT-PCR analyses demonstrated that the RPE-F344 cells
express anti-apoptotic genes associated with cell survival, and several growth
factor receptors important in prostate development and homeostasis. RPE-F344
cells are p27kip1 negative, telomerase positive, and express high molecular
weight cytokeratins speci®c for prostatic basal cells. They also express low levels
of androgen receptor (AR) and prostatic acid phosphatase (PAP); features associated with secretory luminal epithelial cells. RPE-F344 cells are maintained in
vitro without androgen supplementation, but addition of 15nM dihydrotesterone
(DHT) to the culture media results in a signi®cant but transient enhancement of
cellular proliferation. Establishment of RPE-F344-like colonies from rat prostate
is limited to the ventral and dorsal lobes of the prostate 2 ± 4 days after initiation
of regeneration, suggesting that RPE-F344 cells may originate from a stem celllike compartment that is responsible for regenerative repopulation.
Keywords: stem cell; androgen-sensitive; non-transformed
Introduction
The adult prostate in both rats and humans is composed
of stromal and epithelial cells that combine to form a
*Correspondence: GJ Smith, Department of Pathology and
Laboratory Medicine, UNC-Chapel Hill, CB#7525 BBB, Chapel Hill,
NC 27599-7525, USA. E-mail: Cellsort@med.unc.edu
Received 15 June 1999; revised 19 August 1999; accepted
8 September 1999
complex network of secretory glands and connective
tissue. As with other organ systems, much can be learned
about normal biology and the evolution of disease by
isolating and separating the cellular components of the
prostate and culturing them in vitro under controlled
conditions. Mature, secretory epithelial cells can be isolated from normal adult human or rat prostate and
cultured for short periods of time, but long-term maintenance of normal prostatic epithelial cells in vitro has
proven dif®cult. Several prostate cell lines have been
derived from human or rat prostate cancers,1 ± 5 and
Isolation and partial characterization of RPE-F344
SC Presnell et al
258
immortalization with SV-40 Large T antigen has enabled
propagation of quasi-normal prostatic epithelial cells.6 ± 10
However, the immortalized lines do not provide an ideal
model for the study of normal prostate epithelial cells
because they sometimes acquire characteristics of transformed cells such as chromosomal abnormalities, loss of
cell-cycle control, and tumorigenicity.7,11
When rats are castrated, the prostate undergoes involution with consequent loss of the androgen-dependent
secretory epithelial cells.12 ± 15 However, if testosterone is
administered post-castration, the prostate epithelium
undergoes regeneration with re-establishment of normal
tissue architecture.12 ± 15 Although both the basal epithelial cells and secretory epithelial cells are capable of
proliferation, it has been proposed that a stem-like cell
may exist in the adult prostate that is not dependent on
androgen for survival and has the capacity to proliferate
and repopulate the prostate during androgen-stimulated
regeneration.16 ± 19 If such a stem-like cell does exist in the
prostate, its inherent characteristics may make it more
readily propagable in vitro than terminally differentiated
epithelial cells.
In the present study, we sought to establish a prostatic
epithelial cell line from a normal adult male rat without
the aid of immortalizing or transforming agents. By
enriching prostate tissue in vivo for regeneration-competent epithelial cells, we have isolated an androgen-sensitive epithelial cell line, RPE-F344, which is propagable in
vitro, can be cryopreserved reliably, and possesses several
characteristics that are consistent with a less-differentiated or stem-like phenotype. The RPE-F344 cell line
represents a valuable and versatile tool for the study of
prostate biology.
Immunochemical analysis
Immunocytochemical analyses were conducted as
described previously.20 Brie¯y, cells were cultured on
chamber slides, ®xed in 95% ethanol, and subjected to
immunocytochemical staining based on antibody manufacturer's protocols. Primary antibodies included antiAR, anti-Bcl-2, anti-EGFR, anti-c-met, and anti-c-kit (all
from Santa Cruz, Santa Cruz, CA), anti-pancytokeratins
(Sigma, St Louis, MO), anti-keratin 903 (Enzo Diagnostic,
Farmingdale, NY), anti-prostatic acid phosphatase
(DAKO, Carpinteria, CA), and anti-p27kip1 (Transduction Laboratories, Lexington, KY). Proliferation was
assessed in formalin-®xed, paraf®n-embedded rat prostate tissue by immunohistochemical analysis with antibodies to Ki-67 (MIB-5) (Immunotech, Marseille, France),
according to manufacturer's protocol. Secondary antibodies were peroxidase-conjugated goat anti-rabbit or horse
anti-mouse IgG (rat adsorbed). Positive reactions were
visualized with True Blue Peroxidase1 (KPL, Gaithersburg MD) and cells were counterstained with Contrast
Red (KPL). Telomerase activity was assayed with a TRAPeze1 telomerase detection kit (Oncor, Gaithersburg,
MD) using a telomerase-positive rat liver epithelial
stem-like cell line as a control.21
RT-PCR analysis
Messenger RNA was isolated from RPE-F344 cells using
standard methodology.22 Reverse transcription and PCR
were carried out as described previously.20 PCR reactions
were conducted in EasyStart1 PCR reaction tubes (Molecular Bioproducts, San Diego, CA) using primers speci®c
for AR, PAP, TGFbs and TGFb receptors, IGFRs, EGFR,
and c-met. Actin was ampli®ed from each sample to
ensure quality of the reverse transcription and even
loading between samples.
Materials and methods
Generation of the RPE-F344 cell line
Normal three-month old male Fischer-344 rats were
castrated and maintained without androgen for 2
weeks. Testosterone enanthate (0.5 mg) (Schein Pharmaceuticals, Inc., Florham Park, NJ) was administered to
stimulate prostatic regeneration. Four days after administration of testosterone rats were euthanized and the
prostate remnants excised, pooled, digested enzy-matically with 0.5 mg/ml collagenase (Worthington Biochemical Corporation, Lakewood, NJ), and placed in culture in
media supplemented as described previously.20 Prostate
growth media (PGM) consists of Richter's Improved
MEM (Irvine Scienti®c, Santa Ana, CA) supplemented
with 10 mM nicotinamide, 20 ng/ml epi-dermal growth
factor (EGF), 5 mg/ml insulin, 5 mg/ml transferrin, 5 ng/
ml selenium, 100 units/ml penicillin, 100 mg/ml streptomycin, 0.25 mg/ml fungizone and 2% fetal bovin serum or
serum replacement (CPSR-2, Sigma, St Louis, MO). In
some experiments a basal media was used (BPM), which
is PGM without EGF, serum, or phenol red (0.1% bovine
serum albumin (BSA) was in-cluded in BPM to maintain
cell attachment). Epithelial-like colonies were picked and
subcultured to generate clonal cell lines. RPE-F344 were
cryopreserved routinely in PGM containing 15% DMSO.
Growth curve analysis
Tissue culture dishes were seeded with 20000 RPE-F344
cells at passage 14 in supplemented culture media. After
24 hours plates were trypsinized and cell counts obtained
to determine starting numbers. Plates were washed three
times with PBS and re-fed with either (1) PGM, (2) PGM
‡ 15nM dihydrotestosterone (DHT), (3) BPM, or (4) BPM
‡ 15nM DHT. Fresh media was added to plates every 48
hours. Data was plotted and analyzed with Prism 2.01
and InStat 3.0 (GraphPad Software, San Diego, CA).
Flow cytometric analysis
Cultures of RPE-F344 were trypsinized to generate a
single-cell suspension and ®xed by dropwise addition of
cold 95% ethanol, yielding a ®nal suspension of 2 6 106
cells in 70% ethanol. Cells were treated with RNAse
(50 mg/ml) and stained with propidium iodide (20 mg/
ml) at 37 C for 30 minutes immediately prior to ¯ow
cytometry. Chicken red blood cells and a diploid rat liver
epithelial cell line (WB-F344) served as controls for DNA
content. A FACScan ¯ow cytometer (Becton Dickinson
Immunocytometry Systems, San Jose, CA) was used
to evaluate DNA content. Metaphase spreads were
Isolation and partial characterization of RPE-F344
SC Presnell et al
259
generated from the RPE-F344 cell line and from a control
diploid rat liver epithelial cell line (WB-F344), using
standard methodology.23
Evaluation of colony-forming potential of prostate
lobes during regeneration
Nine adult male Fischer-344 rats were castrated via
scrotal incision. Three weeks post-castration 1 mg/kg
testosterone propionate (TP), suspended in sesame oil,
was administered subcutaneously into the subscapular fat
pad at 48-hour intervals. Three rats were sacri®ced at 2
days, 4 days, and 7 days after initial dose of TP. Matched
controls that did not undergo castration or receive TP
injections were included. Additional controls included
rats that were castrated but did not receive TP injections.
At the time of sacri®ce, the remaining prostate tissue was
segregated into anterior, lateral, ventral, and dorsal lobes.
Portions of each lobe from each rat were formalin-®xed
paraf®n-embedded, and processed for immunohistochemistry with anti-Ki67 antibodies (MIB-5) (Immunotech, Marseille, France). Remaining prostate tissue was
pooled according to lobe, enzymatically digested as
described above, and placed in culture in PGM or PGM
‡ 15nM DHT. At 30 days, cultures derived from each lobe
were evaluated by light microscopy, and colonies that
exhibited the morphology of RPE-F344 cells were enumerated. Colonies that could be subcultured successfully
were scored as `RPE-F344-like'.
Results
Isolation and propagation of RPE-F344
Four days after prostatic regeneration was initiated by
testosterone, prostate remnants were harvested and dissociated enzymatically. Isolated cells were cultured in
supplemented media without androgen. After 1 ± 2
weeks several epithelial-like and stromal-like colonies
were chosen and subcultured. One colony gave rise to a
propagable homogenous cell population with a distinct
epithelial morphology. This cell line (designated RPEF344) has been passaged > 50 times and can be cryopreserved reliably in 15% DMSO. The RPE-F344 cells are
shown in Figure 1 at passage 16. The cells form a uniform,
cobblestone monolayer, are contact-inhibited at con¯uence, and do not form colonies in soft agar. Flow cytometric analysis of RPE-F344 at passage 5 showed them
to have a DNA content 12% greater than syngeneic
diploid control WB-F344 rat liver epithelial cells, an
observation that was con®rmed by metaphase chromosome counts where mean chromosome number was 42 in
WB-F344 cells and 48 in RPE-F344.
Figure 1 The RPE-F344 cells are shown at passage 16, grown in PGM.
Photomicrograph was taken on a Zeiss Axiovert 25 at 200 6 magni®cation.
expressed weakly by all cells. The RPE-F344 exhibit
telomerase activity at levels equivalent to a rat liver
epithelial stem-like cell line (WB-F344).21 Additional characteristics of RPE-F344 include strong perinuclear staining
for Bcl-2 and Bcl-XL, expression of HGF receptor (c-met)
on the cell surface and in the cytoplasm, and weak
expression of EGFR and c-kit. Immunocytochemical
analysis with anti-keratin 903 (an antibody speci®c for
high molecular-weight cytokeratins 1,5,10, and 1424 ± 27)
demonstrated strong cytoplasmic and cell surface staining
(Figure 2B), and staining of rat prostate tissue with antikeratin 903 was limited to basal cells (Figure 2C). No
staining was observed in the negative control (Figure 2A).
p27(kip1) protein was not detected in the RPE-F344 cells
by immunohistochemical analysis or Western blot. In
some cases, a few cells within the culture appeared to
be weakly positive for vimentin. Results are summarized
in Table 1.
RT-PCR analysis of RPE-F344 cells
Gene expression patterns of the RPE-F344 cells evaluated
by RT-PCR analysis are summarized in Table 1. Detectable levels of AR and PAP mRNA were present in RPEF344 cells, con®rming immunocytochemical results.
Expression of several growth factors and receptors relevant to the prostate were also evaluated by RT-PCR. RPEF344 cells express mRNA encoding TGFb1, TGFb2,
TGFb3, and both the type 1 and type 2 TGFb receptors.
The cell line also expresses the type 1 and type 2 IGF
receptors, the EGF receptor, and c-met (the receptor for
HGF), but does not produce detectable mRNA encoding
for the ligands for these receptors (IGF1, EGF, TGFa, or
HGF). This is in sharp contrast to a transformed rat
prostate epithelial cell line (HUNC-E) isolated in our
laboratory, which is characterized by autocrine production of several growth factors concomitant with expression of cognate receptors.20
Immunocytochemical analysis of RPE-F344 cells
Con¯uent cultures of RPE-F344 cells grown in the absence
of androgen express immunodetectable levels of PAP and
AR in the cytoplasm (but not in the nucleus). Furthermore, expression of AR and PAP is limited to a proportion
of cells in the con¯uent culture, rather than being
Androgen response of RPE-F344 cells
RPE-F344 cells were evaluated for androgen sensitivity by
evaluation of growth kinetics in BPM or PGM media with
or without 15 nM DHT supplementation. The stimulatory
Isolation and partial characterization of RPE-F344
SC Presnell et al
260
Table 1 Characterization of RPE-F344 by RT-PCR, immuno-histochemistry (IHC), western blot (WB), and TRAPeze1 telomerase
assay (TRAP)
Target
PAP
AR
bcl-XL
bcl-2
P27(kip1)
IGFR1
IGFR2/M6PR
c-kit
c-met
TGFb1, 2, & 3
TGFbRc 1 & 2
Pancytokeratin
Telomerase
CK 1, 5, 10, 14
vimentin
Result
Methodology
7 /weak ‡
7 /weak ‡
‡‡
‡‡
7
‡
‡
weak ‡
‡
‡
‡
‡
‡‡
‡‡‡
7 /weak ‡
IHC/RT-PCR
IHC/RT-PCR
IHC
IHC
IHC/WB
RT-PCR
RT-PCR
IHC
IHC/RT-PCR
RT-PCR
RT-PCR
IHC
TRAP
IHC
IHC
Figure 3 Growth curves were constructed for RPE-F344 cells cultured in
PGM or BPM in the presence or absence of 15nM DHT (A). A signi®cant
increase in cell number at day 5 occurred consistently in both BPM ‡ DHT
and PGM ‡ DHT (B), P < 0.05. Graphs shown are representative of three
separate experiments.
Figure 2 RPE-F344 cells and normal rat prostate tissue were subjected to
immunohistochemical analysis with basal-cell speci®c anti-keratin 903
antibodies. Panel A is a negative control, with no primary antibody.
Panel B is the RPE-F344 cells, stained positively for anti-keratin 903,
and panel C demonstrates that the antibodies are speci®c for basal cells in
normal rat prostate tissue (magni®cation ˆ 200 6 ).
effects of BPM ‡ androgen were evident at day 5, when
the RPE-F344 cultures were expanding rapidly and had
not reached con¯uence (Figure 3A). Growth kinetics of
RPE-F344 were enhanced in PGM compared to BPM,
indicating that the supplements unique to PGM, such as
EGF and serum components, facilitate growth and/or
survival of RPE-F344. Regardless of media formulation
(BPM or PGM), the addition of 15 nM DHT resulted in
a small but signi®cant increase in cell number during
population expansion (Figure 3B), P < 0.05 (one way
ANOVA, Tukey-Kramer multiple comparisons test). The
growth curve and bar graph shown are representative of
three independent experiments. Growth peaked at con¯uence (6 days) in all cultures, and cell numbers
Isolation and partial characterization of RPE-F344
SC Presnell et al
decreased rapidly between days 6 and 8, re¯ecting intolerance of RPE-F344 cells to con¯uent conditions.
Lobe-speci®c potential for establishment of
RPE-F344-like cells
Experiments were conducted to determine whether initiation of prostatic regeneration was necessary for establishment of RPE-F344-like prostatic epithelial cells in vitro.
Lobe-speci®c cultures were established from the prostates
of: (1) intact adult male rats; (2) adult male rats three
weeks post-castration; (3) and adult male rats three weeks
post-castration ‡ 2, 4, and 7 days of testosterone-initiated
regeneration. After 30 days of culture, no epithelial colonies with RPE-F344-like morphology were identi®ed in
cultures derived from intact or 3-week castrate rats.
Colonies with a morphology indistinguishable from
RPE-F344 were identi®ed only in cultures derived from
regenerating ventral and dorsal prostate, at both 2 days
and 4 days (but not 7 days) after initiation of regeneration
(Table 2). Neither the anterior nor lateral prostate lobes
gave rise to RPE-F344-like colonies under any conditions.
No propagable epithelial colonies were derived from any
cultures that included DHT as a media supplement.
Immunohistochemical analysis of tissues harvested
from the prostate tissues demonstrated that testosterone-induced proliferation was limited to the dorsal and
ventral lobes of the regenerating rat prostate. Furthermore, proliferation peaked at 2 and 4 days, and returned
to low levels by 7 days after the initiation of regeneration
(Figure 4). Interestingly, the peak of proliferation in the
regenerating ventral and dorsal prostate (2 ± 4 days) coincides with the optimal time points for establishment of
epithelial colonies in vitro.
Table 2 Colony-forming potential of each lobe of the prostate in
intact rats and in rats that were subjected to one cycle of prostatic
involution/regeneration, 2, 4, and 7 days after administration of
1 mg/kg testosterone propionate
Lobe
Anterior
Lateral
Dorsal
Ventral
Intact
2-day Regen.
4-day Regen.
7-day Regen.
0
0
0
0
0
0
3
4
0
0
4
2
0
0
0
0
Figure 4 Proliferative activity was identi®ed by immunohistochemical
detection of the Ki-67 nuclear antigen in dorsal and ventral prostates of
intact adult male rats and rats that underwent testicular castration followed
by 2, 4, and 7 days of testosterone-induced regeneration. Positive cells are
identi®ed by a dark blue nuclear stain (magni®cation ˆ 200 6 ).
Discussion
The hypothesis that a stem-like cell exists in the prostate
that can give rise to mature prostatic secretory epithelial
cells has been investigated since the demonstration that
the involuted prostate of a castrated rat regenerates fully
upon treatment with androgen.12 A stem cell model of the
prostate has been proposed by Bonkhoff and Remberger
in which: (1) the three basic cell types encountered in the
prostatic epithelium Ð i.e. secretory luminal, basal, and
endocrine ± paracrine cells Ð are linked in a precursor ±
progeny relationship; (2) the regenerative compartment of
the normal and hyperplastic epithelium is located in the
basal cell layer; and (3) the stem-cell compartment is
androgen-independent for survival but contains androgen-responsive target cells involved in repopulation
during regeneration.28 Verhagen et al used basal and
luminal cell-type speci®c cytokeratins to demonstrate
that the basal cell compartment in the regenerating rat
prostate contains cells that proliferate upon androgen
administration and give rise to intermediate cell types
that possess markers for both basal and luminal epithelial
cells.28 The prostatic basal cell compartment in situ has
been characterized as androgen-independent but androgen-responsive,27 expressing little or no nuclear AR,29 ± 31
no PSA,32,33 and large amounts of high molecular weight
cytokeratins, such as those detected by the anti-keratin
903 antibodies.34,35 In contrast, mature luminal epithelial
cells require androgen for maintenance and express high
levels of AR, PSA, and cytokeratins 8 and 18.19,34,35 De
Marzo et al have described a stem cell model of the
prostate in which highly proliferative stem cells from
the basal compartment give rise to a population
of partially differentiated transiently proliferating cells,
which then give rise to the post-mitotic secretory epithelial cells.19
The goal of the present study was to isolate normal rat
prostatic epithelial cells that could be propagated in vitro
without the introduction of immortalizing genes, to serve
as a normal counterpart to transformed rat prostate cell
lines, such as the HUNC-E derived from the Dunning rat
prostate cancer model.20 The RPE-F344 cells were isolated
from involuted prostatic remnants four days after administration of exogenous testosterone; a time when the
purported stem-like cells are proliferative and a signi®cant number of transitional cells are present.27 While ®ve
epithelial-like colonies were selected from the original
culture, only one colony gave rise to an easily propagable
cell line that retained epithelial morphology and characteristics. The RPE-F344 cells were not immortalized with
SV40 or other potentially transforming agents, but have
been passaged in vitro > 50 times. The cells were originally established in de®ned media containing 2% FBS,
which does contain some androgen. However, 0.1% BSA
can replace FBS in the culture media, indicating that the
trace amounts of androgen present in FBS do not contribute to survival of the cell line. Evaluation of growth
kinetics with or without exogenous androgen demonstrated a transient increase in cell number in androgen
treated cultures during population expansion, indicating
that the RPE-F344 cell line is mildly androgen-responsive.
During growth curve analysis, all cultures (regardless of
media formulation) achieved peak density at day 6, with
261
Isolation and partial characterization of RPE-F344
SC Presnell et al
262
a rapid decline in cell number during days 7 ± 10, re¯ecting contact inhibition and an apparent intolerance to
sustained con¯uent culture conditions. Immunocytochemical and RT-PCR analyses demonstrated expression
by the RPE-F344 cells of several genes associated with cell
growth and survival: Bcl-2, Bcl-XL, c-kit, and several
growth factor receptors important in prostate development and homeostasis (c-met, EGFR, TGFbRs). Anti-keratin 903 antibodies (speci®c for cytokeratins 1, 5, 10, and
14), which decorate only basal epithelial cells in situ in
normal prostate tissue, stained the RPE-F344 cells
strongly in vitro, providing evidence that the RPE-F344
cells were derived from the basal cell compartment.
Expression of AR and PAP was cytoplasmic and weaker
in RPE-F344 than in a well-differentiated rat prostate
adenocarcinoma cell line (HUNC-E) established in our
laboratory,20 suggesting a less differentiated phenotype.
Characteristics of the prostate stem cell population, as
de®ned by DeMarzo et al, include expression of telomerase and Bcl-2, and lack of expression of p27kip1.19 Our
data suggest that the RPE-F344 cell line, which is telomerase ( ‡ ), Bcl-2 ( ‡ ), Keratin 903 ( ‡ ), and p27kip1 ( 7 ),
may represent the proliferative basal compartmentderived stem cells that are capable of differentiating to a
more mature phenotype in an appropriate microenvironment. This hypothesis is supported by the observation
that establishment of RPE-F344-like epithelial cells in
culture is limited to dorsal and ventral prostate 2 ± 4
days after initiation of regeneration, i.e. only regeneration-competent lobes of the prostate give rise to RPEF344-like cells, and only during the time of maximum
proliferation.
In summary, the RPE-F344 cell line represents a unique,
normal prostatic epithelial cell line with a stem-like
phenotype that has not been genetically modi®ed to
achieve immortalization but can be maintained in vitro
for extended periods of time. The RPE-F344 cells can be
used to conduct studies of gene expression, differentiation, and malignant transformation in vitro and in vivo,
using techniques such as transfection, cell tagging, and
orthotopic/ectopic transplantation. Furthermore, the
observation that establishment of rat prostate epithelial
cell lines was limited to prostate tissue during the peak of
tes-tosterone-induced regeneration suggests that a similar
strategy may aid in the successful isolation of non-transformed human prostate epithelial cell lines.
Acknowledgements
This work is supported by NIH-CA64865.
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