[CANCER RESEARCH 49, 3050-3056, June 1, 1989]
Transformation of Human Neonatal Prostate Epithelial Cells by Strontium
Phosphate Transfection with a Plasmid Containing SV40
Early Region Genes
M. Edward Kaighn,1 Roger R. Reddel,2 John F. Lechner, Donna M. Peehl, Richard F. Camalier, Douglas E. Brash,
Umberto Saffiotti, and Curtis C. Harris
Laboratory of Human Carcinogenesis [M. E. K., R. R. R., J. F. I., D. E. B., C. C. H.] and Laboratory of Experimental Pathology [U. SJ, Division of Cancer Etiology,
National Cancer Institute, Bethesda, Maryland 20892; Developmental Therapeutics Program [R. F. C.], Division of Cancer Treatment, National Cancer Institute,
Frederick, Maryland 20701; and Division of Urology [D. M. P.], Stanford University Medical Center, Stanford, California 94305-5118
ABSTRACT
Neonatal human prostatic epithelial cells (NP-2s) were transfected by
strontium phosphate coprecipitation with a plasmid (pRSV-T) containing
the SV40 early region genes. The cells transfected with pRSV-T, but not
the sham-transfected controls, formed rapidly growing, multilayered col
onies within 2 weeks at a frequency of 1 x 10 4 in a serum-free medium
(P4-8F).
In all, 28 colonies of transformed cells were isolated. Three of
these have been cultured for a sufficient length of time to show that their
growth potentials are well beyond that of the normal progenitor cells
(NP-2s). There is also little or no indication of the culture "crisis"
commonly seen in SV40-transformed cells in these transfected lines. All
contain cytokeratins and SV40 T-antigen as revealed by immunofluorescence, have ultrastructural features of epithelial cells, and are pseudodiploid. None have produced tumors within 1 year after s.c. injection into
nude mice. The transformed as well as the parental NP-2s cells require
bovine pituitary extract for growth in serum-free medium and are stimu
lated by transforming growth factor #, (TGV-ß,)and epidermal growth
factor in clonal growth assays. In contrast, a prostatic carcinoma cell line
(PC-3) is inhibited by TGF-/Õ,. This serum-free system and immortalized
transfected clones will be useful for studying the action of putative
prostatic carcinogens and tumor-promoting agents.
quences. Since calcium phosphate was toxic to NP-2s cells, we
substituted the newly developed strontium phosphate transfec
tion procedure.
Transformed cells are generally less dependent upon and less
responsive to mitogenic factors than are their normal counter
parts. Earlier, we found that NP-2s cells infected with SV40
virus had reduced mitogen requirements in serum-supple
mented medium (2). During the past several years, the com
plexity of the role of hormones and growth factors in controlling
both growth and differentiation pathways has received more
attention. In particular, TGF-/3i has been shown to have bifunctional regulatory activities, the nature of which varies with
cell type and the total cellular environment (8). The availability
of a serum-free medium simplified the study of the effect of
TGF-01 and other factors on the growth of NP-2s cells and its
T-antigen gene-transfected derivatives (9).
MATERIALS AND METHODS
Cell Culture. Culture procedures were as previously reported (2)
except that a serum-free growth medium (P4-8F) was used (10). This
consisted of P4, a modified basal medium PFMR4 (11) without the
trace element concentrate and with the Ca2+ concentration reduced to
INTRODUCTION
In 1978, methods suitable for the isolation of replicative
epithelial cells from human neonatal prostate were described
(1). These cells, NP-2s, were later transformed by infection with
SV403 virus (2). Several colonies with extended culture lifespans, chromosomal aberrations, reduced growth factor de
pendence, altered morphology, and anchorage-independent
growth were isolated. However, they were not tumorigenic, nor
did they develop into established "immortal" cell lines. Further
attempts to induce immortality and tumorigenicity by infection
or transfection by ras oncogenes, or by exposure to chemical
carcinogens were unsuccessful (3).
Renewed interest in utilizing the NP-2s system for investi
gating the etiology of prostatic cancer was stimulated by recent
technological advances in culturing human prostate cells (4, 5)
and in introducing cloned oncogenes into human epithelial cells
(6). Because of the difficulties in inducing tumorigenicity by
SV40 infection, possibly due to the fact that a small fraction of
cells remain capable of viral synthesis (7), we explored the use
of transfection to introduce only the SV40-transforming seReceived 9/21/88; revised 2/17/89; accepted 2/27/89.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
' To whom correspondence and requests for reprints should be addressed.
1 Present address: Children's Medical Research Foundation, P. O. Box 61,
Camperdown, N. S. W. 2050, Australia.
3 The abbreviations used are: SV40, simian virus 40; TGF- 0i, transforming
growth factor TGF- ßt;
BPE, bovine pituitary extract; PD, population doublings;
HBS, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic
acid buffered saline; EGTA,
ethyleneglycol bis(/3-aminoethyl ether)-Ar,Ar,Ar',A"-tetraacetic acid; SSC, standard
saline citrate (0.15 M NaCl-0.15 M sodium citrate, pH 7.0).
0.5 HIM(Biological Research Faculty & Facility, Inc., Ijamsville, MD).
P4-8F consisted of P4 basal medium supplemented with 30 nM H2SeO3
(Johnson Matthey Chemicals, Ltd., Hertfordshire, England), 5 ng/ml
epidermal growth factor (Collaborative Research), 5 Mg/ml insulin, 0.28
JIMhydrocortisone, 0.5 UMphosphoethanolamine, 0.1 DMcholera toxin
(Calbiochem), 250 fig/ml bovine serum albumin (Sigma), and 0.5%
BPE prepared in this laboratory as previously described (12). TGF-/3i
was obtained from R & D Systems, Inc., Minneapolis, MN. Culture
dish surfaces were treated with a coating mixture [100 ng/m\ bovine
serum albumin, 10 Mg/m' bovine fibronectin (both from Calbiochem)
and 20 ¿ig/mltype 1 collagen (Vitrogen 100, Collagen Corp., Palo Alto,
CA)] to promote attachment (13). PC-3, a prostatic carcinoma line
(14), was also cultured in serum-free P4-8F. Adult human prostate cells
were cultured as previously described (4, 5). Transfected cell lines,
which were difficult to dissociate, were suspended by a sequential
procedure. Either culture dishes were washed with HBS, then treated
for 15 min at 37°C.with K-pass Solution A which contains (in g/liter):
KC1, 8.4; NaCl, 7.1; NaH2PO4 •
7H2O, 1.9; KH2PO4, 0.005; polyvinylpyrrolidone (Calbiochem), 10; glucose, 3.4; and 4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid, 4.8, pH 7.4. Serum which was present
in the original Solution A (1, 11), was omitted. Solution A was removed
and the cells were suspended with PET [1% polyvinylpyrrolidone,
0.02% EGTA, 0.02% crystalline trypsin (Sigma) in HBS, pH 7.4] at
room temperature. When required, 0.4 mg/ml deoxyribonuclease-I was
added to further aid dissociation. Cultures were found to be free of
mycoplasma by R. A. Del Giudice, Program Resources, Inc., NCIFCRF, Frederick, MD (15).
Transfection. The plasmid, pRSV-T obtained from Dr. B. Howard,
National Cancer Institute, is an SV40 ori~ construct containing the
SV40 early region genes and the Rous sarcoma virus long terminal
repeat (6). NP-2s cells were recovered from frozen stocks, subcultured
once, and were plated at the 25th PD level (1-2 x 106/100-mm dish)
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PROSTATE EPITHELIAL
CELL IMMORTALIZATION
BY SV40 GENES
in 10 ml of growth medium P4-8F. The plasmid DNA was coprecipitated with strontium phosphate as previously reported using the mod
ification described in the addendum of Reference 6. After 4-h treatment,
the precipitate was removed, and the cells were shocked by addition of
1 ml of 15% glycerol in HBS (w/v) for 30 s. The monolayer was washed
three times with basal medium (P4), and incubated in P4-8F medium.
Five days later, the plates were subcultured (2 x 10s cells/100-mm
2s cells. However, colonies of transformed cells that developed
in cultures transfected with pRSV-T by the strontium phos
phate technique could be seen by phase microscopy within 2
weeks and had reached a diameter of about 1 cm by 4-8 weeks.
The transformation frequency in three separate experiments
was approximately 1 x 1(T4. No colonies developed in control
dish). The culture medium was replaced three times per week.
Isolation of Transformed Colonies. Colonies of tightly packed, multilayered cells which reached a diameter of 1 cm or more within 4-8
weeks were observed on dishes containing pRSV-T-transfected cells,
but not on the control dishes. Colonies were ringed with glass cylinders
and individually dissociated by sequential treatment as described in
"Materials and Methods." The cells were cultured and expanded in P4-
dishes containing only the strontium phosphate precipitate
(Fig. 1) or constructs carrying H-ras, v-myc, H-ras + v-myc, or
8F medium. The ability of the transfected cells to form colonies on a
background of senescing normal cells was the only selective procedure
used.
Clonal Growth Assays. Subconfluent cultures were suspended with
PET and plated at 500 cells/60-mm dish in P4-8F; four dishes per
variable were used. After overnight incubation, the medium was re
placed with experimental media. After 6-7 days' incubation, plates
were fixed with 10% buffered formalin and stained with Giemsa. Cells/
colony were counted using an image analyzer. The mean clonal growth
rate (PD/day) was based on counts of at least 18 colonies (2).
Tumorigenicity Assay. Cells were suspended with PET and injected
s.c. in the mid-dorsal craniad region of athymic nude mice (riunii)
(NCI-FCRF, Frederick, MD). A total of 2 x IO6 cells in 0.1 ml of
medium were used per animal. At least five animals per cell line were
injected. Similar protocols were also used at Stanford (D.M.P.), but in
addition one animal per cell line was used for histological examination
of the inoculum site at 2 weeks. Animals were observed for tumor
development up to 12 months.
Chromosomal Analysis. Karyotypic analyses were carried out by Dr.
Ward D. Peterson, Jr., Children's Hospital of Michigan, Detroit, MI,
by Giemsa banding (16). Exact chromosome counts of 30 metaphases
per line were made for ploidy determination. For each culture, six to
10 karyotypes were prepared.
Immunostaining and Electron Microscopy. Cytokeratins and SV40 I
antigen were identified by indirect immunofluorescence as previously
described (17). Cells were grown on coated Chamber-Tek (Miles) slides
and fixed with acetone at —20°C.
Mouse monoclonal antibodies were
used to detect large and small T-antigens (Oncogene Science, Mineóla,
NY), and cytokeratins (Labsystems, Chicago, IL). Primary antibodies
were subsequently stained with goat fluorescent anti-mouse ¡ninnino
globulin antibody. Additional immunoperoxidase labeling was carried
out at Stanford (D.M.P.) using an avidin-biotin-peroxidase complex
(18). Cells were prepared for transmission and scanning electron mi
croscopy by standard procedures (17).
Southern Analysis. Cell lines were analyzed for the presence of
integrated SV40 early region DNA. Genomic DNA (10 jig) was digested
with restriction enzymes BamHl (which cleaves a single site in pRSVT) and Xbal (to which pRSV-T is resistant). The DNA digests were
electrophoresed on 1% agarose gel, and the DNA was transferred (19)
to Gene Screen Plus membrane (New England Nuclear, Boston, MA).
Membranes were prehybridized in 1% sodium dodecyl sulphate/1 M
NaCl/10% dextran sulfate for 6 h at 65°C.The blots were then
hybridized with "P-labeled DNA probe overnight at 65°Cin prehybridization solution containing 200 Mg/ml denatured salmon sperm DNA.
The probe, labeled by nick translation (20) was the 1.2-kilobase Hindill
DNA fragment of the SV40 early region from plasmid pRSV-T. The
membranes were washed twice for 5 min with 2 x SSC at room
temperature, twice with 2 x SSC/1% sodium dodecyl sulfate for 30
min at 65°C,and twice with 0.1 x SSC for 30 min at room temperature.
The membranes were autoradiographed
-70°C.
• •
Fig. 1. Transformation of normal human prostate epithelial cells by SV40
early region genes. Photomicrographs of cultures of NP-2s cells fixed and stained
5 weeks after transfection with the plasmid pRSV-T. No colonies were visible on
the control plates (top). Tightly packed multilayered colonies of variable size
formed on the pRSV-T-treated plates (bottom).
i•
.0f
O.</
nya3o
°/V'o°•
'
oCo'£ti"3aoa.100-,80604020n.-/
o• ,A
«OmÄ '
,AQ•
AO..•
267B1J-j)»
<«,.
272E1-F^
¿£A
272A91
• •
o
A
o
A
with an intensifying screen at
i
O
i
30
!
!
,
60
Days
RESULTS
Transfection and Isolation of Transformed Cells. Preliminary
experiments showed that calcium phosphate was toxic to NP-
•
90
;
1
!
120
150
i
(-
180
-:
!
210
*
--
240
of Incubation
Fig. 2. Cumulative growth of three transformed prostatic epithelial cell lines.
Normal cells (NP-2s) were transfected at time 0. Each line was isolated from a
single colony. The population doublings have not been corrected for plating
efficiency at each passage. 267 and 272, two independent experiments.
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PROSTATE EPITHELIAL CELL IMMORTALIZATION
BY SV40 GENES
Fig. 3. Electron micrographs of a trans
formed prostatic epithelial cell line (267B1).
Transmission electron micrograph (16,500 x).
The scanning micrograph (inset, 500 x) shows
abundant surface microvilli and rounded divid
ing cells.
mr-
i*
the adenovirus 12 genes Eia and Elb. Initially, the colonies
consisted of small aggregates that developed into tightly packed,
multilayered colonies that were very difficult to reduce to a
single-cell suspension. However, sequential treatment with a
solution containing an elevated K+ concentration, followed by
trypsinization and addition of deoxyribonuclease was effective
in reducing the extent of clumping. The reduced ability of the
transfected cells to attach to the culture dish surface was elim
inated by coating the surface of the dishes ("Materials and
Methods").
Escape from Senescence. The cumulative growth of three
selected transformed lines is shown in Fig. 2. Two lines, 267B1
and 272E1, are approaching the 100th PD, whereas 272A9 has
reached the 65th PD level. Except for a lag period in the case
of 272E1, no indication of a traditional crisis has been seen. In
contrast, the life expectancy of the normal cells at transfection
was approximately 10-15 PDs (1).
Characterization. The transformed colonies had the typical
polygonal arrangement of epithelial cells by phase contrast
microscopy (not shown). Transmission and scanning electron
micrographs (Fig. 3) revealed features comparable to those
originally reported for NP-2s (1,11). Analyses of eight isozymes
were carried out on NP-2s, 267B1, 272E1, and 272E4. All had
the human lactic dehydrogenase and type B glucose-6-phosphodehydrogenase. These data support the conclusion that the
transfected lines are derived from NP-2s (16). Cytokeratins 8
(A/r 52,000) and 18 (M, 45,000), vimentin, actin, and fibronectin
were demonstrated in all three transfected cultures by indirect
immunoperoxidase staining (Table 1). Intense nuclear staining
was observed in all three transfected cultures with anti-SV40
T-antigen, but not in normal cells. In many respects, the label
ing pattern was similar to that of adult prostate epithelial cells.
Labeling of structural filaments was less intense in the trans
fected lines. In 272E4, some cells were intensely stained
whereas other were negative with anticytokeratin 8. Cytokera
tins 1,5, 10, and 11 stained intensely in the adult cells but not
in the transfected lines (not shown). In cell line 272E4, keratin
8 is strongly expressed in denser cultures. In some cases antisera
to a single antigen from different sources gave variable results.
This was true of prostatic acid phosphatase, prostatic specific
antigen (Table 1), collagen Type IV, and laminin (not shown).
Factor VIII, an endothelial cell antigen, was not detected.
Chromosomal Alterations. Karyotypic analysis of several
transformed lines revealed varying mixtures of normal and
aneuploid populations. All lines had the Y-chromosome. The
chromosome counts of four lines are summarized in Table 2.
In most cases, 90% of the chromosome counts were in the
diploid range (2n = 46). The line 267B1 (36 PDs) consisted
mostly of normal male cells with random loss and gain of
normal chromosomes. However, by the 53rd PD level, 267B1
had become 24% tetraploid and had a significantly higher
proportion of abnormal metaphases, monosomy, trisomy, re
arrangements, dicentrics, and gaps than were observed at 36
PDs. Cell lines 272E1 and 272E4 had normal as well as
aneuploid cells. 272E1 had a higher proportion of aneuploid
cells than did 272E4. Fig. 4 shows an abnormal 46 chromosome
karyotype of 272E1 at the 36th PD level carrying a 4p+
translocation and an aberrant chromosome 14. No consistent
losses, gains, or stable marker chromosomes were seen. In
contrast, the parental line, NP-2s was predominantly normal
male (XY) diploid even when approaching senescence at the
30th PD level. Two metaphases, one with a missing chromo
some 12, the other with a missing chromosome 9 were seen,
indicating an occasional lack of division fidelity as the normal
NP-2s culture approached senescence (2). No marker or unas
signable chromosomes were found.
Tumorigenicity. None of the transfected lines have formed
tumors in nude mice 12 months after s.c. inoculation. This
observation was confirmed at Stanford (D.M.P.) and at North
western University by Dr. E. W. Sherwood.4 Dr. Sherwood also
' Personal communication.
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PROSTATE EPITHELIAL CELL IMMORTALIZATION
It
Fig. 4. Karyotype of transformed line,
272E1, at the 20th PD. A 4p+ translocation
(arrow) and an aberrant no. 4 chromosome are
shown.
BY SV40 GENES
It
8
M
ti
ft*
it
15
21
Table 1 Antigenic markers in transfected prostatic epithelial cells
Cells were fixed and stained as described in "Materials and Methods." Labeling
was assigned values from - (none) to +++ (very intense).
Cell culture
Antigen
Keratin 8*
Keratin 18e
Vimentin1'
Actin'
Fibronectin-^
Factor VIII*
SV40 T-antigen*
PAP;
PSA'
267B1
272E1
272E4
±
±
±
±
±
±
Adult
" Heterogeneous staining.
* Enzo Biochemicals mouse antikeratin 8, 1:2000.
' Boehringer Mannheim mouse antikeratin 18, 1:50.
''Enzo Biochemicals mouse antivimentin, 1:200.
' Miles rabbit antiactin, 1:40.
•^ICN
mouse antifibronectin, 1:200.
' ICN goat anti-factor VIII, 1:200.
* Oncogen mouse anti-SV40 T-antigen, 1:1000; nuclear staining only.
' Prostatic acid phosphatase was negative with Ortho rabbit anti-PAP, undi
luted and positive with Miles rabbit anti-PAP, 1:300.
' Prostatic specific antigen was negative with Ortho rabbit anti-PSA, undiluted
and positive with aberrant nuclear staining with Dako rabbit anti-PSA, 1:400.
Table 2 Chromosome numbers of normal and transfected prostatic epithelial cells
Data represent the number of metaphases in the specified range. 100 metaphases per culture were scored.
CellcultureNP-2s doubling
level-SO
267B1
267B1272E1
diploid(42-48)*86 tetraploid
(85-92)IIe
36
5325
92
824
76
92
8
272E4Population
25Near
91Near
9
°Estimated population doublings since isolation (NP-2s) and since transfection
ofNP-2s.
* Range of chromosome numbers.
' This analysis contained three of 100 metaphases with >150 chromosomes.
scanty pale cytoplasm sometimes forming acinar structures.
There were frequent mitoses and evidence of infiltration into
the surrounding stromal, blood vessels, and muscle tissue. The
histological pattern was consistent with carcinoma in all three
lines. Observation at subsequent times showed that the nodules
never increased in size and eventually regressed. Line 272E4
showed more irregular nuclear sizes and shapes and evidence
of pyknosis.
Response to Growth Factors. TGF-01 has been reported to
inhibit growth or to stimulate terminal differentiation in epi
thelial cells (21). The effect of TGF-/3, on the growth rate of
the transformed lines was tested by clonal growth assay (Fig.
5). For comparison the parental line, NP-2s, and the prostatic
carcinoma line, PC-3, were included. TGF-ßistimulated the
growth rate of the normal as well as the transformed lines, but
inhibited the growth of PC-3 at high levels. Bovine pituitary
extract was found to be both essential for serum-free growth
and, at higher levels, slightly inhibitory (Fig. 6).
Southern Analysis. For each of three cell lines analyzed for
the presence of SV40 early region DNA, hybridization to the
T-antigen gene probe revealed single bands in genomic DNA
digested with either Xbal (for which there is no restriction site
in pRSV-T) or BamHl (which cleaves pRSV-T once outside of
the sequence corresponding to the probe) (Fig. 7). There was
unequally intense hybridization to the probe. However, inspec
tion of the gel after electrophoresis by ethidium bromide stain
ing showed a corresponding inequality of DNA loading, and
prolonged autoradiography revealed no additional bands (not
shown). It is therefore likely the pRSV-T has integrated at only
one site in each of these three cell lines. The SV40 probe did
not hybridize with the control DNA from PC-3 cells.
DISCUSSION
observed no tumors following intrasplenic inoculation. The
histology of the inocula at the s.c. injection sites was examined
2 weeks after injection. In all three lines, small nodules, 2-5
mm in diameter, were observed. They were formed by clusters
of cells with largely mostly oval nuclei, prominent nucleoli and
Prostatic cancer, now the third most prevalent form of cancer
in men and the second leading cause of male cancer-related
deaths, is becoming more significant as life expectancy is ex
tended (22). Despite the dimensions of this problem, little is
known about the etiology of prostatic carcinogenesis (23). The
main purpose of this work was to develop a culture model
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PROSTATE EPITHELIAL
CELL IMMORTALIZATION
BY SV40 GENES
Bam H1
PC-3
Xba I
r
267B1
A 272E4, P8
0.8
10
Xba I
A272E1, P7
100
Bam H1
Bam H1
IODO 10000272 A9
Xba I
TGF-ftlpg/ml)
Bam H1
272E1
Xba I
g
.1
3
I
I
P
O>
CO
j*
7;
o-
Fig. 7. Southern analysis of SV40 early region DNA in transformed prostatic
epithelial lines. The probe used was the 1.2-kilobase Hindlll DNA fragment of
the SV40 early region.
I
0.5
1.0
100
10
1000
10000
(6) yielded stabile transfectants with an efficiency of 1-2 x
10~4. This frequency of transformation is approximately the
TGF-p, (pg/ml)
Fig. 5. Effect of TGF- ß,on clonal growth of transformed human prostatic
epithelial cells. Top, three pRSV-T-transfected lines, 267B1, 272E4, and 272E1;
bottom, normal prostate line, NP-2s, and a human prostatic carcinoma line, PC3.
120 T
100
g
U
~a
i
during which the rate of proliferation markedly decreases or
ceases entirely (26). More recent reports indicate that crisis
does not always occur and its incidence and severity may vary
with cell type, for example, human keratinocytes often become
immortalized without the intervention of crisis (26).
Three other types of human epithelial cells have been transfected with pRSV-T. Four of four clonally derived transfected
bronchial lines senesced (21), and two survived crisis and be
came immortalized.6 Similarly, four pRSV-T-transformed
80
60
-
c
o
g
0>
PU
40-
20
O
0.00
same with both the calcium and strontium procedures providing
that the cells can withstand both. The observed transformation
frequency is identical to that achieved in this laboratory using
the same plasmid with human cells from the bronchial epithe
lium (21), nicsothelium (24), and esophagus.5
All types of normal human cells appear to have finite lifespans in culture (25). Human cells infected with SV40 virus
have extended lifespans but, in most cases, undergo a "crisis"
0.25
0.50
0.75
1.00
BPE (%)
Fig. 6. Response of the transformed line, 267B1, to BPE. Cells were plated
in medium P4-8F minus BPE. After 18 h, the medium was replaced with
experimental medium. Clonal cultures were stained after 7 days and colonies of
more than 20 cells were counted. Data points are percentage of maximum CFE
(0.25%). Error bars, SD.
suitable for investigating the etiology and progression of pros
tatic cancer. Since normal prostatic epithelial cells have a short
lifespan, it was important to find a way of extending their
replicative potential without introducing genes allowing viral
synthesis. Efforts to introduce the SV40 early region genes into
NP-2s by the standard calcium phosphate transfection method
were unsuccessful due to the toxicity of the calcium phosphate.
However, the newly developed strontium phosphate procedure
mesothelial lines senesced and only one recovered from crisis
(24). Two pRSV-T-transfected esophageal lines also have sur
vived crisis.5 In contrast, the 3 pRSV-T-transfected NP-2s lines
that have been in culture long enough to permit a prediction of
their growth potential appear to have become immortalized
without a crisis, with only a short (approximately 40-day) lag
occurring in the 272E1 population. Whether immortalization
without crisis is a general characteristic of prostatic epithelial
cells transfected with pRSV-T awaits further study. In the
previous study where NP-2s cells were infected with SV40 virus,
all of the transformed isolates had senesced by the 45th PD (2)
and none developed into immortal lines. It is unclear whether
the contrasting result obtained in the present study is due to
the different method of introducing the SV40 early region and
5 G. D. Stoner, unpublished.
6 R. R. Reddel el al., unpublished.
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PROSTATE EPITHELIAL
CELL IMMORTALIZATION
the absence of late region genes, or due to the substantially
improved culture techniques used in the present study.
The transformed cultures express T-antigen by immunoperoxidase nuclear staining. This is consistent with the Southern
blotting data indicating that each line contains a single copy of
integrated SV40 early region DNA. The antigenic profile of the
transfected lines is quite similar to that of the adult prostatic
epithelial cells (Table 1). They express cytokeratins as expected
for cells of epithelial origin. All three lines are similar to benign
prostatic adenomas and have nearly identical two-dimensional
gel electrophoresis patterns of [35S]methionine-labeled cell ex
tracts. They contain acidic (no. 19, Mr 40,000) and basic (no.
6, M, 56,000) cytokeratins.7
Although the finding that TGF- ß\stimulated the growth
rate of the normal NP-2s and of T-antigen-transfected cells
might appear in contrast with the known inhibitory effect of
TGF- ß,on several types of epithelial cells (27-30), TGF- ß,is
not always inhibitory for epithelial cells (8). Human mesothelial
cells are also stimulated by TGF- ßi(31). Since NP-2s, its
SV40-transformed sublines, and the mesothelial cells were iso
lated and cultured in media containing serum, it might be
expected that these lines could grow in the presence of TGF/3i, a factor present in serum (27). The prostatic carcinoma line
(PC-3) was inhibited by TGF- ß\
confirming a report concerning
the androgen-independent prostatic cancer lines, PC-3 and DU
145 (32). Furthermore, the closely related TGF- ß2
has been
isolated and purified from PC-3-conditioned medium (33). The
mechanism by which normal prostatic cells are stimulated by
TGF- ßt,whereas neoplastic cells are inhibited is unknown.
However, these findings suggest that an autocrine mechanism
of growth control may be involved. The availability of a serumfree system and the present T-antigen gene-immortalized lines
will facilitate investigation of this hypothesis.
The karyotypic alterations in the transfected prostatic epithe
lial cells are similar to those previously reported after infection
with SV40 virus (2). They are also comparable to those in the
immortalized human bronchial epithelial line, BEAS-2B (21).
Most transfected lines were near diploid with random loss and
gain of chromosomes. With continued culture, an increase in
chromosome number and other aberrations developed. Insta
bility of the karyotype also appears to be a characteristic of
virally transformed cells (26, 34).
None of the tested transfected lines were tumorigenic in nude
mice, although a transient growth of carcinoma-like cells was
observed at 2 weeks. Stanbridge et al. (34) have formulated a
model for the progression of a normal to a neoplastic cell via a
transformed but nontumorigenic intermediate. The transfected
lines may represent such an intermediate in which altered
growth control is offset by the capability of undergoing differ
entiation when injected into mice. Others have also reported
that T-antigen immortalized human and other primate cells are
nontumorigenic in nude mice (35-37). Recently, isolation of a
nontumorigenic human bladder line, immortalized by SV40
infection and selected by the ability to escape senescence, has
been reported (38). These cells have subsequently been con
verted to tumorigenicity by treatment with 3-methylcholanthrene. In view of the lack of knowledge relating to prostatic
carcinogenesis, the immortalized prostatic lines described
herein will also be useful for studying the effects of putative
carcinogens and tumor-promoting agents.
7 E. W. Sherwood, personal communication.
BY SV40 GENES
ACKNOWLEDGMENTS
The authors thank Dr. Bruce Howard for plasmids, Dr. Ward Peter
son, Jr., Children's Hospital of Michigan, for isozyme analysis and
karyotyping and K. Kelly, V. Sasser, and E. Pauls for expert technical
assistance. We are grateful to Dr. E. W. Sherwood, Northwestern
University Medical School, for unpublished data on the transfected
lines.
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Transformation of Human Neonatal Prostate Epithelial Cells by
Strontium Phosphate Transfection with a Plasmid Containing
SV40 Early Region Genes
M. Edward Kaighn, Roger R. Reddel, John F. Lechner, et al.
Cancer Res 1989;49:3050-3056.
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