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Re v i e w A r t i c l e OGH Reports
Non-human prostate cancer cell lines
Shantibhusan Senapati,1* Subhankar Chakraborty,2 Indramani Nath,3 Susen K Panda,4
Ramesh C Patra,5 Surinder K Batra6
Institute of Life Sciences, Nalco Square, Bhubaneswar, Orissa 751023, India
Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68198, USA
3
Department of Surgery and Radiology, College of Veterinary Science and Animal Husbandry, Orissa University of
Agriculture and Technology, Bhubaneswar 751003, India
4
Department of Veterinary Pathology, College of Veterinary Science and Animal Husbandry, Orissa University of Agriculture
and Technology, Bhubaneswar 751003, India
5
Department of Veterinary Medicine, College of Veterinary Science and Animal Husbandry, Orissa University of Agriculture
and Technology, Bhubaneswar 751003, India
6
Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198, USA
1
2
ABSTRACT
Cell lines have always been a valuable tool to address a variety of questions related to prostate cancer. Due to
the unavailability of many human prostate cancer cell line models, investigators have shown significant interest
in developing or establishing different new nonhuman prostate cancer cell lines. Indeed, many animal cell line
models have successfully recapitulated key events in human prostate cancer development. For instance, Dunning
rat prostate cancer cell lines have created a system to represent the full spectrum of prostate cancer progression
while canine cell lines like Leo and Ace-1 recapitulate bone metastasis model. The establishment of in vitro
models of animal cell lines recapitulating human disease will aid in molecular and functional characterization of
human prostate cancer.
Keywords: Cancer, prostate, canine, mouse, cell line, model, human, rat.
INTRODUCTION
Prostate cancer (PCa) is a multistep disease. It progresses
from prostatic intra-epithelial neoplasia (PIN) to locally
invasive adenocarcinoma, which further leads to hormonerefractory metastatic carcinoma.[1–4] Therefore, an understanding of the molecular changes that initiate and/or
promote prostate cancer progression is essential to design
any therapeutic strategy for this disease. Though different
animal models have contributed significantly in this regard,
*Corresponding address:
Shantibhusan Senapati, B.V.Sc, Ph.D.
Ramalingaswami Fellow Institute of Life Sciences
Nalco Square Bhubaneswar 751023 India
Office: 0091 6742300137/2301476
Email: [email protected], [email protected]
DOI: 10.5530/ogh.2012.1.5
Oncol. Gastroenterol. Hepatol. Reports Vol.1 / Issue 1 / Jul–Dec, 2012
none have been able to mimic all the steps of prostate
cancer progression. Animal models have other limitations
including ethical concerns and high management costs.
Therefore, to understand prostate cancer pathogenesis
in an effective manner, other avenues must be adopted.
One such avenue is the use of cell culture models to study
the process of cancer development and progression in an
in vitro environment. Furthermore, cell line models also
have importance in developing novel therapeutic agents.
Importantly, cell culture provides a system that can be easily manipulated experimentally, and enables evaluation of
response to a wide range of stimuli on a single cell.
Despite the high incidence of prostate cancer in men,
there are only a few cell lines of human prostate carcinoma available. The cell lines LNCaP, PC-3 and DU-145
cells, and some clonal derivatives of these cell lines are
still the most commonly used cells for the majority of
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Shantibhusan Senapati, et al.: Non-human prostate cancer cell lines
published research. Information obtained from these cell
lines and their sub-lines, while valuable, has limitations
and has led to a continued gap in our information regarding molecular basis of prostate cancer pathogenesis. In
prostate cancer research, the development of many new
animal cell line models has been of great use to answer
questions unique to PCa. These cell lines are either established from primary tumor tissues or clonal derivatives of
already established cell lines. Aside from humans, dogs
and a few strains of rats develop spontaneous prostate
cancer.[3,5,6] Use of carcinogens also induces prostate cancer in rats.[7] Moreover, some genetically engineered mouse
models also develop spontaneous ­prostate cancer.[1] In
recent years, there has been a growing effort towards isolating and establishing animal cell lines from these models. Indeed, many of these animal prostate cancer cell line
models have been successfully used to mimic different
events of prostate cancer progression and metastasis.
Immense efforts have been made to catalogue the entire array
of human PCa cell lines earlier.[8,9] However, less attention
has been paid in discussing the information about cell lines
obtained from non-human species (mostly from rodents and
canines). Therefore, we have made an effort to review all the
available information regarding different animals PCa cells’
method of origin, characterization and use here.
Molecular markers for rodent and canine prostate
epithelial cells
Prostate cancer is a multistep process.[3] Therefore, a
thorough understanding of the parental lineage of different established cell lines is crucial before their use in
different functional studies. In human beings, rodents
and dogs, the normal prostate epithelium is characterized
by the presence of three distinct types of cells: luminal,
basal and neuroendocrine (Fig. 1).[10,11] The luminal cells
are often considered terminally differentiated; and secretory and basal cells are thought to be poorly differentiated.
Identification of different markers has been successfully
used to distinguish different prostate cell lines as per
their lineage of origin. Prostate epithelial cells of different lineages express specific intermediate filament (IF)
proteins, cytokeratins (CKs). The luminal cells express
low molecular weight cytokeratins CK8 and CK18 as the
typical marker.[10–12] They also produce different secretory
proteins such as prostate specific antigen (PSA) in men,
and arginine esterase in dogs.[13] Luminal cells also express
androgen receptor. Basal cells of humans and mice prostate epithelium express high molecular weight cytokeratins
CK5 and CK14.[11] However, in dogs CK5 expression is
abundant and CK14 expression is limited and ­scattered.[12]
16
Figure 1. A generalized diagram showing specific markers for
different type of prostate cells.
Different investigators have also identified other basal cell
specific markers such as Ki67, p63, CD44, ETS-2, glutathione S-transferase P and Maspin.[11,14,15] These cells rarely
express androgen receptors (AR).[15] In normal prostate,
Neuroendocrine (NE) cells express both luminal and
basal cytokeratins (CK 5, 14, 8 and 18), and the neuroendocrine marker, chromogranin A.[11,16] However, in cancer,
the NE cells express chromogranin A and only luminal
­cytokeratins.[17] The NE cells do not express androgen
receptor and PSA.[11,18] In addition to the aforementioned
three cell types, another intermediate or transition cell type
is found in prostate epithelium, which expresses both the
basal and luminal CK cell markers, but does not express
AR and PSA.[19] Experimental evidence has suggested that
Prostate stem cell antigen (PSCA) and CD44 are two potential markers for intermediate prostate epithelial cells.[20,21]
Canine prostate cancer cell lines
Dogs are the only lager mammals other than humans,
which commonly suffer from spontaneous prostate ­cancer.
Prostate cancer in dogs has many important similarities to
prostate cancer in humans.[6] Therefore, canine prostate
cancer is a relevant model to understand prostate cancer
pathogenesis. Cell lines derived from spontaneous canine
prostate cancer have been successfully used for in vitro and
in vivo studies. Prostate cancer in dogs is highly invasive and
metastasizes to distant organs.[6] Particularly, the ability of
many canine prostate cancer cells to metastasize to the bone
has created a lot of interest leading to different researches
in this field. However, unlike human prostate cancer cells,
the canine prostate cancer cells are androgen independent
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Shantibhusan Senapati, et al.: Non-human prostate cancer cell lines
from the very early stages of the cancer. Therefore, most of
the established canine prostate cancer cell lines are believed
to recapitulate only the late stage (androgen independent)
of human prostate cancer progression.
Leo
Thomas J Rosol’s group from the Ohio State University
has established and characterized two important canine
prostate cancer cell lines, Ace-1 and Leo. The Leo cell
line was generated from a primary spontaneous tumor
obtained from a five-year-old castrated male mixed breed
dog.[22] The cells were tested positive for CK8 and CK18,
thus indicating their secretory epithelial origin. These cells
also express ductal cell marker CK7, which suggests that
they have differentiated towards both secretory and ductal types.[12] Leo cells also express arginine esterase.
Genomic analysis of Leo cells showed chromosomal
imbalances such as hyperdiploidy and bi-armed chromosomes.[22] In standard cell culture, these cells grow in an
anchorage-dependent manner and form a monolayer sheet
with a tightly packed cobblestone growth pattern. The cells
have a doubling time of approximately 38 hours. Subcutaneous injection of ten million Leo cells into nude mice
generated tumors of an average size of 0.8 cm3 in 6 weeks.
Tumors were multilobular and the microscopic appearance
of subcutaneous xenograft cells was similar to the primary
tumor.[22] Furthermore, the authors have also checked the
metastatic ability of these cells by left ventricular intracardiac injections. Intra cardiac injection of Leo cells tagged
with YFP-Luc resulted in metastasis to different organs.
Out of 25 male nude mice, 23 mice were detected with
metastasis.[22] After 28 days of injection, histopathological
analysis of the different organs obtained from these mice
showed the presence of metastasis in brain, spinal cord,
adrenal gland, long bones and scapula, and the incidence of
brain metastasis was very common (76% mice). The bone
metastasis that was detected in athymic (mice deficient in
T-cell immunity) mice was predominantly osteolytic type.
This cell line is particularly valuable because it repeatedly
develops brain metastasis in vivo; therefore, it will have
­significant contribution in brain metastasis study.
Ace-1
Ace-1 cell line was established from Ace-1 xenografts in
nude mice.[23] Originally, the authors obtained prostate carcinoma tissue from an 8-year-old male castrated Labrador
Retriever, and processed and injected them ­subcutaneously
into nude mice. The Ace-1 xenograft was then serially passed
through implantation into new nude mice. The cell line was
developed from 3–5 passages of the Ace-1 xenografts.
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For this, tumors were minced, collagenase-digested, and a
single-cell suspension was prepared by using a 70 μM filter.
To suppress stromal cell growth, the cells were grown in
either DMEM/F12 media or Keratinocyte serum-free
media. The Ace-1 cells were noted to express CK 8/18, but
not CK 5/14. These cells also express the mesenchymal
marker vimentin. The authors felt that the expression of
vimentin in these cells might be due to epithelial to mesenchymal transition (EMT). These cells are negative for AR.
Ace-1 cells are locally invasive and metastasize to the bone
in nude mice. The bone lesions were of a mixed osteoblastic and osteolytic reaction similar to bone metastasis in
human patients with prostate cancer.[23] Therefore, this cell
line is a potential tool for studies related to bone metastasis.
DPC-1
This cell line was established from a spontaneous
canine prostate adenocarcinoma.[24] The primary prostate cancer tissue was obtained from an 11-year-old
male Doberman Pinscher. After mechanical dissociation
and mincing, tumor fragments were plated in supplemented ­RPMI-1640 medium. In culture, the cells have
rapid growth kinetics with an average doubling time of
27 hr; however they failed to produce colonies in soft
agar suggesting their inability to grow in an anchorage
independent manner. The cells have epithelial morphology in culture, and expressed epithelial-specific CK19.
An inconsistent expression of CK14 and vimentin has
also been reported. No androgen receptor expression
was detected.[24] The karyotype of DPC-1 cells was hypoploid with a chromosome number ranging from 67 to 70
(78 is normal). DPC-1 cells are tumorigenic in both athymic mice and immunosupressed dogs.[24,25] Subcutaneous injection of these cells in nude mice showed a 100%
tumor uptake, and no gross metastasis was detected by
the authors. Histologically, the tumor cells were highly
undifferentiated prostate carcinoma. Orthotopic allograft
in immunosupressed dog produced a poorly differentiated primary tumor.[25] In this system, metastasis to adjacent lymph nodes and lung was detected.
CPA-1
This cell line was established from a spontaneous canine
prostate tumor acquired from a castrated 10-year-old
Doberman dog.[26] Small fragments of the original tumor
tissues were washed with PBS and trypsinised for 45 min.
The mixture of tissue fragments and single cells was filtered and the filtrate was centrifuged. Further, the cell
pellets were resuspended in DME medium supplemented
with 5% FBS. Initially, the tumor cells obtained from the
primary tumor were grown in semisolid agar. Individual
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Shantibhusan Senapati, et al.: Non-human prostate cancer cell lines
colonies grown in the agar were aspirated and grown in
separate wells of a 24-well cell culture plate. Out of all
the isolated clones, the rapidly amplifying clones were
­further sub-cultured. CPA-1 cell line was generated by this
method. In culture, CPA-1 cells have an epithelial growth
pattern. At low-density, these cells form a monolayer; however, high density culture forms multi layering of cells.[26]
In xenografts obtained from CPA-1, cells have a similar
morphology that is found in primary tumor implants.
Androgen and estrogen receptors were undetectable in
the homogenates of the primary tumor tissue and cell cultures. Moreover, the growth of these cells was insensitive
to andogen or estrogen in vitro, and was similar in both
sexes of mice in vivo. These cells are tumorigenic in athymic mice, and tumors were of well differentiated type.[26]
CT- 1258
This cell line was established from a spontaneous prostate tumor obtained from a 10-year-old male intact ­Briard
dog.[27,28] The tumor was removed by surgical method,
minced, collagenase-treated, and dissociated cells were
cultured in medium 199. The cells are tumorigenic in
NOD/Scid mice.[27] The authors have reported 89% of
tumor incidence when these cells were injected subcutaneously into male and female nude mice. Histopathological analysis showed no metastasis in any of these animals.
Histologically, the subcutaneous tumors showed a highly
heterogeneous population of cells with different genetic
abnormalities. Immunohistochemistry showed a strong
Ki67 staining (Ki 67 index 41%) indicating a high proliferative nature of these tumor cells. CT-1258 cells also
have the ability to develop tumors when injected inraperitonially, and in this case 86% of the animals developed
local tumor growth at the injection site. In this system the
authors did not observe any metastasis. Importantly, the
experimental tumors induced in mice (both subcutaneous and intraperitoneally) have a similar growth pattern
and histological appearance as the original tumor from
which the cell line was established.[27] Cytogenetic analysis of the original tumor, the cell line and experimentally
induced tumors showed the presence of hyperdiploid
­karyotypes.[27,28] Furthermore, centromeric fusion between
chromosomes 1 and 5 and chromosomes 4 and 5 were
also detected. There was also presence of a large biarmed
marker (mar).[27] These findings support the thought that
human and dog prostate cancer have the same molecular
events in the generation and/or progression of cancer.
a­ dvancements in science have made it possible to generate
genetically engineered mouse models (GEMs) that generate prostate cancer.[1] Due to the spontaneous (without
carcinogen) and autochthonous development of prostate
cancer in an intact immunity set up, these models have
been instrumental in understanding the genetic basis of
prostate cancer initiation and progression. Furthermore,
different cell lines established from these models have
been helpful in different prostate cancer studies. Use of
many of the cell lines in xenograft model has also generated potential tools in the prostate cancer therapeutic
studies.[29–31]
TRAMP-C1/C2/C3
These cell lines were established from a 32 week old heterogeneous prostate tumor obtained from a transgenic
adenocarcinoma mouse prostate (TRAMP) model.[32]
TRAMP model is a transgenic mouse on a C57BL/
6 background, which carries a construct that expresses SV40
large T antigen in prostate epithelium.[33] After differential
trypsinization to get rid of stromal cell contamination, the
cells were maintained in supplemented DMEM-HG media.
These cell lines were positive for cytokeratin, E-cadherin
and androgen receptor.[32] Immunohistochemical analysis
also suggested the absence of mutated p53 in these cells.
As reported by Watson AP et al., TRAMP-C1 cells particularly express CK14, CK8, Psca, and Sca-1 markers.[34]
Out of these markers, Sca-1 is considered to be a marker
for cells with stem cell properties. Therefore, TRAMP cells
might have originated from intermediate or stem cell like
cells. In soft agar assay, TRAMP-C1 cell had very few colonies after 10 days of growth, while TRAMP-C2 did not
form any colonies.[32] Ironically, the three cells lines do not
express SV40 T ­antigen.[32]
The TRAMP-C1 and TRAMP-C2 cells are tumorigenic
in syngeneic C57BL/6 mice, and tumor uptake was 100%
in both cells. However, TRAMP-C3 which grew very well
in vitro, failed to form any tumor in syngenic C57BL/6
mice.[32] Histological analysis of TRAMP-C1 and
TRAMP-C2 xenograft tumors revealed poorly differentiated and disorganized sheets of cells. By using serial prostatic implantation of TRAMP-C1 cells, Somers KD et al.
have established a metastatic tumor cell line; TRAMPC1P3.[35] Orthotopic implantation of T
­RAMP-C1P3
cells in C57BL/6 mice leads to predominant lymph node
metastasis.
Mouse prostate cancer cell lines
Prostate neuroendocrine cancer (PNEC) cell lines
Unlike dog and a few strains of rat, normal mice do
not develop spontaneous prostate cancer. However,
Prostate neuroendocrine cancer (PNEC) cell lines were
generated from 20 to 24-week-old FVB/N transgenic
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Shantibhusan Senapati, et al.: Non-human prostate cancer cell lines
mice (CR2-Tag) primary tumors and metastases.[36]
In these mice, simian virus 40 large T antigen (Tag) is
expressed in a subset of prostate NE cells.[37] When cells
obtained from different organs were cultured in supplemented neural progenitor medium (NPMM), all of them
grew with a similar morphology. PNEC30, PNEC25 and
PNEC28 cells were derived from primary tumor, liver
metastasis and lymph node metastasis, respectively. All
these cells grow in suspension as aggregates on a non
coated surface; however, they attach to poly (L-lysine) and
human laminin-coated surfaces and grow as monolayer.
The average doubling time of these cells is 50 h, and they
produce neurite-like process in culture. A subline named
PNEC30-3 grows as a monolayer on polystyrene surfaces.
In suspension, the aggregation formation of these cells
resembles the neurospheres of cultured neural stem cells.
Subcutaneous injection of PNEC30 cells in BALBc mice
produced tumor in all the recipients and the incidence
was independent of sex. Furthermore, on tail vein injection, liver tumors were identified in the injected mice.
GeneChiP analysis indicated that PNEC cells express
steady features ex vivo and in vivo. Cell lines harvested at
different passages (30 or 34), and xenografts share a high
degree of similarity with the primary CR2-Tag prostate
tumors. PNECs cells express mAsh1, a basic helix-loop
helix (bHLH) transcription factor in a cell cycle dependent manner. mAsh1 transcription factor is known to
be essential for NE cell differentiation.[38] Based on their
phenotypes, PNEC cells are a valuable tool to address
questions associated with NE cells proliferation, differentiation and tumorigenesis.
E and cE series (E2, E4, cE1, and cE2)
All these four cell lines were established from tumor tissues obtained from different conditionally Pten deleted
mice (cPten-/-L).[39,40] E2 and E4 cell lines were derived
from androgen dependent PCa, and cE1 and cE2 cell
lines were obtained from androgen-depletion independent (ADI-Ca) prostate tumor. Androgen dependent cells
were obtained from an intact male mouse 8 months of
age, and ADI-Ca cells were obtained from the prostate
tumor of a 13 month-old male mouse, that was castrated
at the age of 8 months.[39] In culture, these cells have
mostly had polygonal shaped morphology and possess a
bi-allelic deletion of Pten gene locus, and all of them have
a similar level of androgen receptor (AR) expression.
Karyotyping analysis showed that E2, 4 and cE2 are near
tetraploid, and cE1 was aneuploid. E2 and E4 cell lines
express low level of CK5 and p53 (basal cell markers)
and no luminal markers, and were androgen dependant
for their optimal growth in culture. On the other hand
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cE1 and cE2 cells express both luminal (CK8 and CK18)
and basal cell markers (CK14). Like E2 and E4 cell lines
cE1 and cE2 also express a low level of CK5. They are
capable of growing in an androgen independent manner
in vitro. Under deprivation of androgen, cE1 and cE2 cells
express more AR and proliferate when exogenous androgen is provided. Further gene expression analysis showed
that, E2 and E4 cells express higher level of genes that are
considered to be EMT markers (vimentin, Twist, Slug and
Snail) than cE1 and cE2 cells.[39]
These two series of cell lines differ in their tumorinducing abilities in vivo. E2, E4 cells when injected into
NOD-SCID mice, formed sarcomatoid carcinoma in a
sex independent manner. However, cE series cell lines
formed adenocarcinoma only in males and undifferentiated carcinoma in the females.[39] These cell lines are
thought to be potential tools to understand the prostate
cancer progression.
PTEN-P2, PTEN-P8, PTEN-CaP2 and PTEN-CaP8
PTEN-P2 and PTEN-P8 cell lines were originated
from the prostate of a 10 month old Ptenloxp/loxp; ­PB-cre4+
mouse.[41,42] Out of several clonal derived cells, PTENP2 and PTEN-P8 cell lines were two independent
spontaneously immortalized lines. Genotyping of
PTEN-P2 and PTEN-P8 cells revealed that both the
cells are genetically heterozygous for Pten deletion.
Therefore, to generate isogenic lines with homozygous
deletion of Pten allele, the authors infected PTEN-P2
and PTEN-P8 cells with Cre-retrovirus.[41] The PTENCaP2 and PTEN-CaP8 isogenic lines were generated
after 2 weeks of puromycin drug selection. PCRbased genotyping and westernblot analysis confirmed
the homozygous deletion of Pten in these cell lines,
and this was further corroborated by the detection of
increased P-AKT level in these cells. Pten deletion also
increased the level of AR receptors, supporting a previous finding by these authors that PTEN negatively
regulates AR expression.[43] Q-RT-PCR analysis showed
that these cells express both basal (CK14) and luminal (CK8/CK18) cytokeratins. However, they do not
express the neuroendocrine markers. Further detection of PSCA expression (an intermediate cell marker)
in these cells suggested that these cells have probably
originated from a transiently amplified or intermediate
cell type.[20] The cytogenetic analysis of all the cell lines
showed several common chromosomal abnormalities
indicating their common origin. Importantly, PTEN-P8
and PTEN-CaP8 showed the loss of Y chromosome, a
frequently found cytogenetic change that is observed in
human sporadic prostate tumors.[44]
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Shantibhusan Senapati, et al.: Non-human prostate cancer cell lines
Additionally, in the soft agar assay (anchorage independent growth) PTEN-CaP2 and PTEN-CaP8 cells formed
more and bigger colonies compared to ­PTEN-P2 and
PTEN-P8 cells.[41] Furthermore, compare to isogenic
heterozygous pairs, Pten null cells have a decreased level
of p27 and E-cadherin expression. After subcutaneous
injection, Pten null cells were able to generate visible
tumors; however, in a similar condition the heterozygous cells did not form any tumor. Histological analysis
showed maintenance of AR and CK8 expression in all
the xenografts, and no mesenchymal marker (SMA) and
no neuroendocrine makers were detected in any of the
xenografts.[41]
Myc-CaP
This cell line was established from a 16-month-old
­Hi-Myc transgenic mouse.[34,45] The cells were maintained in a growth factor enriched/supplemented
DMEM media. Myc-CaP cells express c-Myc as equivalent to the level seen in the original prostate carcinoma
tissue.[34] These cells also overexpress AR, which might
be due to genomic amplification of AR as detected
by FISH analysis. However, the authors were unable
to detect any mutation in AR after sequencing of all
the exons. Myc-CaP cells express CK14, CK18, Psca
and Sca-1. This expression profile of different lineage
markers suggests that these cells might originate from
an intermediate or stem cell type. Additionally, soft agar
assay in the presence or absence of androgen revealed
that these cells are androgen dependent for their growth
and/or survival.
Myc-CaP cells are tumorigenic when injected in the mammary pad of syngenic male mice.[34] In this study, the
authors have reported a 100% tumor uptake. ­Histologically,
the xenografts uniformly composed of undifferentiated
cells. To check the effect of androgen on the tumorigenicity of these cells, the authors castrated the tumor
bearing mice and measured the tumor volume at multiple
time points. Interestingly, though Myc-CaP xenofrafts
regressed initially, later on they grew in an androgen independent ­manner.[34] As this cell line has been derived from
a primary prostate tumor that has never been exposed to
hormone deprivation, it is believed to be a prospective
reagent for understanding the development of hormone
refractory prostate cancer.
177-2 and 178-2 series cell lines
These cell lines were established from primary and
metaststic tumors of mice that carry prostate tumor
through a mouse prostate reconstitution (MPR)
20
model.[46] Firstly, urogenital sinus epithelial cells
(UGE) of different p53 genotype were grafter under the
renal capsule of adult male syngeneic 129 mice (p53 +/
+). These mice were then provided with a special diet
without or with synthetic retinoids N-(4-hydroxyphenyl)
retinamide (4-HPR). After several days, when the animals were with obvious distress, the primary and metastatic tumors were harvested and evaluated. 178-2PA cell
lines were obtained from the primary tumors of the animals on control diet, and 178-2 BMA, BMB and BMG
cell lines were obtained from the bone metastatic tissue of the animal on control diet. Likewise, 177-2 PB/
PE cell line was derived from primary tumor of animals
with 4-HPR diet and 177-2 BMA cell line was derived
from bone metastasis. 178-2 BMA cells also have the
capacity to form primary and metastatic (lung) tumors
in syngenic orthotopic mouse model. The orthotopic
model of this cells line has been extensively used for
many prostate cancer therapeutic studies.[31,47]
Rat prostate cancer cell lines
Like humans and dogs, some strains of rats also develop
spontaneous prostate cancer.[5,7] Therefore, spontaneous prostate cancer in rats has created an opportunity
to isolate and establish different cell lines that represent
­different stages of prostate cancer progression. Moreover, carcinogen-induced prostate cancer model is a
well established model and has been used to address
many prostate cancer associated questions.[48] Investigators have also shown interest to generate different cell
lines from these carcinogen-induced prostate cancer
tumors.
Dunning cell lines (G, AT1-3, MAT-LyLu, and MAT-Lu)
This series of cell lines were derived from Dunning rat
prostate cancer sublines (in vivo tumors), which have different biological characteristics.[49] The original tumor
from which all the in vivo sublines were established
was R3327 tumor (discovered by W.F. Dunning) in a
22-month-old inbred Copenhagen male rat.[50] Further
by serial in vivo passages of the R3327 tumor, various
tumor sublines (G, AT1-3, MAT-LyLu and MAT-Lu)
were generated. And these tumor lines have difference
in their histology, androgen sensitivity, growth rate and
metastatic ability. To establish the in vitro cell lines, after
collagen digestion, filtration and plating, the confluent
primary culture cells were continuously passaged. During
the culture of these cell lines, glucocorticoid hormone
supplementation was observed to be critically required
for the attachment of growing cells to the plastic surface.[49] Particularly, AT3 and MAT-LyLu cell lines were
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Shantibhusan Senapati, et al.: Non-human prostate cancer cell lines
more susceptible to detachment in absence of glucocorticoid in the culture medium.
Different analysis confirmed that all the Dunning in vitro
cell lines have retained the major properties of their
­relevant parental tumor sublines.[49] For example, G cell
line has a slower growth rate and AT1-3, MAT-LyLu and
MAT-Lu have a faster growth rate in vitro. To characterize these cell lines in vivo, when these lines were injected
subcutaneously back into castrated and intact Copenhagen male rats, all the aforementioned cell lines produced
100% tumor uptake.[49] Like its parental tumor subline,
G cells growth was androgen sensitive (grew better in
intact than castrated rats) and was low metastatic. All
other cell lines like their parental tumor line (AT1-3,
MAT-LyLu, and MAT-Lu) grew in an androgen insensitive manner (growth rate was same in both intact and castrated rat). AT-3, MAT-Lu and MAT-LyLu cell lines were
highly metastatic. AT3 and MAT-LyLu cell lines metastasized to lymph node and lung. As well as MAT-Lu cell line
metastasized to lung. However, AT-1 and AT-2 have very
low metastatic ability.
MDR lines (AT3B-1, AT3B-2, MLLB-1, and MLLB-2)
MDR lines were developed by culturing the parental
AT-3 and MAT-Ly-Lu (MLL) cell lines in increasing
concentration of doxorubicin.[51] These cell lines were
maintained in 1 μM doxorubicin for more than one year.
In contrast to parental cell lines, the MDR lines were
more resistant to vinblastine treatment. Like their parental cell line (AT3), AT3B-1 and AT3B-2 cells developed
tumor when subcutaneously injected in Copenhagen
rats.[51] Interestingly, xenografts produced by these two
MDR lines were less responsive to doxorubicin treatment than AT3-xenografts in vivo. Overexpression of
P-glycoprotein has been thought to contribute to the
MDR phenotypes of these cells.[51] These cell lines are
very good model to study chemotherapy resistance in
prostate cancer.
NRP152 and NRP154 cell lines
In the year 1994, Danielpour A et al. had generated
these two cell lines from the dorso-lateral prostate of
two Lobund/Wistar rats (rat number 152 and 154)
treated with N-methyl-N-nitrosourea and testosterone
­propionate.[52] To develop NRP152 cell line, dorsolateral prostate from carcinogen and hormone treated
animals was excised, minced, and the fragments were
plated on tissue culture flasks. After cells attached to
the plates, the cells were treated with trypsin-EDTA to
remove ­fibroblasts from the cultured cells. After serial
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t­rypsinization and passages, one clone that remained
attached to the culture plate and continued to grow was
developed into NRP152 cell line. With some modifications, NRP-154 cell line was also generated in a similar
fashion like NRP152 cell lines. After harvesting prostate
tissue, prostate tissues were digested with collagenase,
passed through a filter, and sequentially washed with
WAJC 404 with and without FBS. Further, cells were
cultured in a special serum free media (SFM) that inhibits stroma cells growth. After some days, the medium
was replaced with serum containing media.[52] Finally
one clone that survived and continued to grow was
established as NRP-154 cell line.
In culture, both the cell lines have different morphology.
At a low density NRP-152 cell monolayers are cuboidal, but sporadically aligned in parallel curved bundles
at a high density. Compare to NRP-152 cells, NRP-154
cells are larger and flatter. The NRP-152 cells have many
properties similar to normal prostatic epithelial cells.
They express functional androgen receptor and prostatic
acid phosphatase. On the other hand, NRP-154 cells are
malignant type; and the level of AR m-RNA is undetectable in these cells.[52] For optimal growth, in addition to
10% FBS NRP-152 cells need additional growth factors
and hormones; in contrast, only 10% FBS is sufficient for
NRP-154 cells growth. Addition of TGF-β to the culture
media inhibits growth of both the cell lines. However,
retinoic acid inhibits growth of only NRP-152 cells, and
has no overall effect on growth pattern of NRP-154 cells.
Under optimal growth conditions, both the cell lines have
a doubling time about 17 h. NRP-152 cells were unable to
form tumors in athymic mice (in the presence and absence
of matrigel). However, NRP-1524 cells are tumorigenic
in athymic mice.[52] Though k-ras mutation is a common
finding in MNU-induced prostate cancer, neither of these
cell lines has this mutation.
CONCLUSION AND FUTURE DIRECTIONS
Advancement in cell line models has been instrumental in understanding the pathogenesis of prostate cancer. ­However, certain issues associated with cell culture
have always generated doubts on many cell line based
data. Amongst them, misidentification and cross-contamination of different cell lines are the biggest hurdle
in cell-culture based studies.[53] Therefore, investigators
working with different cell lines should take personal
moral responsibility to make sure that no cell lines are
infected with any microorganisms like mycoplasma, and
the genetic identity of different cell lines are routinely
21
Shantibhusan Senapati, et al.: Non-human prostate cancer cell lines
Table 1 Relevant information about different animal prostate cancer cell lines
Name of the
cell line
DPC-1
Ace-1
Leo
CT 1258
CPA-1
cE1
cE2
E2
E4
Myc-CaP
178-2 BMA
PTEN-P2
PTEN-P8
PTEN-CaP2
PTEN-CaP8
TRAMP-C1
TRAMP-C2
TRAMP-C3
PNEC 30
G
AT-1
AT-2
AT-3
MATLy-Lu
MAT-Lu
NRP 152
NRP 154
AT3B1, B2
Species
Dog
Dog
Dog
Dog
Dog
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
AR
expression
No
No
No
NA
No
Yes
Yes
Yes
Yes
Yes
NA
Yes
Yes
Yes
Yes
Yes
Yes
Yes
NA
Yes
No
No
No
No
Specific cytokeratin
expression
14, 19
8, 18
8,18,7
NA
NA
5,8,14,18
5,8,14,18
5
5
14,18
NA
8,14,18
8,14,18
8,14,18
8,14,18
14,8
NA
NA
NA
NA
NA
NA
NA
NA
No
Yes
No
NA
NA
14
18
NA
checked. During ­multiple passages, additional genetic
changes may accumulate in different cancer cells, which
might have an impact on the morphology and phenotypes of those cells. Therefore, detection of any kind of
such alterations should be well documented. It is also
important to check the characteristic features of tumors
that are obtained from ­different cell lines, and should
be compared to the original tumor phenotypes. During
tumorigenesis study, more information about the host
animals (species, strain, age, and sex) will be very instrumental in using these systems in a more clinically relevant
manner.
Like human cancer cell lines, most of the aforementioned animal cancer cells are derived from already
transformed malignant cells. Therefore, these cells
are not suitable to understand the early genetic events
that initiate or ­promote prostate cancer. Generation of
more cell lines from different new GEMs that develop
spontaneous tumor is essential to understand the mechanism of prostate cancer initiation and promotion. Cell
lines with known genetic alterations will be also helpful inscreening the efficacy of drugs with a particular
22
Tumorigenisity
Metastasis
Reference
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
No
NA
NA
NA
NA
NA
NA
Yes
NA
NA
NA
NA
NA
NA
NA
NA
Yes
Yes
Yes
Yes
Yes
24, 25
23
22
27, 28
26
39, 40
39, 40
39, 40
39, 40
34
46, 47
41, 42
41, 42
41, 42
41, 42
32, 34
32
32
36
49
49
49
49
51
No
Yes
Yes
Yes
NA
NA
NA
51
52
52
49
genetic back ground. Later, information obtained
from these models can be translated to personalized
human cancer therapy. Due to the origin of spontaneous prostate cancer in dogs, and its biological similarity
with human prostate cancer, canine prostate cancer in
vivo and in vitro models have a significant importance
in the prostate cancer research. Importantly, preliminary evaluation of the canine genome advocates a more
resemblance between dog and human than human and
rodents in term of nucleotide divergence and rearrangements. Moreover, comparative genomic studies have
shown significant homology between different cancerrelated genes like MET, mTOR, KIT and IGFR1R both
in dog and humans.[54] Therefore, generation and characterization of more canine prostate cancer cell lines
will be helpful to the whole prostate cancer research
community.
ACKNOWLEDGEMENTS
S.S. is a recipient of Ramalingaswami Re-entry Fellowship
of Department of Biotechnology, GOI, New Delhi.
Oncol. Gastroenterol. Hepatol. Reports Vol.1 / Issue 1 / Jul–Dec, 2012
Shantibhusan Senapati, et al.: Non-human prostate cancer cell lines
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