[CANCER RESEARCH 46, 3630-3636, July 1986]
Tissue Culture Model of Transitional Cell Carcinoma: Characterization of
Twenty-two Human Urothelial Cell Lines
John R. W. Masters,1 Peter J. Hepburn, Lawrence Walker, Wilma J. Highman, Ludwik K. Trejdosiewicz,
Susan Povey, Mohamed Parkar, Bridget T. Hill, Peter R. Riddle, and Leonard M. Franks
Department ofHistopathology, St. Paul's Hospital, Institute of Urology, 24 Endell Street, London WC2H 9AE, United Kingdom [P. J. H., J. R. W. M., L. W., W. J. H.,
L. K. T.]; Gallon Laboratory, M. R. C. Human Biochemical Genetics Unit, University College, 4 Stephenson Way, London NWl 2HE, United Kingdom [S. P., M. P.];
and Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, United Kingdom [B. T. H., P. R. R., L. M. F.J
ABSTRACT
Twenty-two continuous cell lines derived from normal and neoplastic
urothelium, maintained under identical culture conditions, were charac
terized in terms of isozyme phenotype, tumorigenicity, and xenograft
morphology following xenotransplantation to nude mice, cylological ap
pearance, in vitro growth rate, labelling index, and colony-forming effi
ciency, in parallel with separate studies of in vitro drug sensitivities and
monoclonal antibody reactivities. Three groups were identified: (a) dis
tinct lines with differing isozyme patterns, a broad spectrum of growth
characteristics, and xenograft morphologies similar to the histopathology
of the parent tumors after periods of up to 17 yr following establishment
in vitro; (b) cross-contaminated sublines (maintained separately in differ
ent laboratories for periods of up to 10 yr), with identical isozyme patterns
and similar growth characteristics, but differing markedly in tumorigenic
ity and xenograft morphology; and (c) lines derived from normal uro
thelium which were nontumorigenic and had an isozyme pattern usually
only encountered in untransformed cells. These data indicate that cell
lines representative of human transitional cell carcinomas can be selected
on the basis of xenograft morphology and isozyme patterns, and that a
panel of lines derived from normal and neoplastic urothelium could
provide a model system to study the biology and treatment of this disease.
COLO232 (Professor G. E. Moore, Denver General Hospital, Denver,
CO); HT1197 and HT1376 (Professor S. Rasheed, University of South
ern California, Los Angeles, CA); HU456, HU609, and HU961T (Dr.
M. Vilien, Fibiger Laboratory, Copenhagen, Denmark); MGH-U1 and
MGH-U2 (Professor G. R. Prout, Massachusetts General Hospital,
Boston, MA); PS1 (Dr. E. J. Sanford, Pennsylvania State University,
State College, PA); KK47 (Professor H. I lisa/unii. Kanazawa Univer
sity, Kanazawa, Japan); HS0767 (Dr. W. A. Nelson-Rees, Naval Biosciences Laboratory, Berkeley, CA); TCCSUP, SCaBER, J82COT, and
T24 (Dr. C. O'Toole, Cambridge, United Kingdom); and HCV29 (Dr.
J. Fogh).
Cell Culture. All lines were grown routinely on plastic as monolayers
in 25-cm2 flasks (Nunc; Gibco, Paisley, Scotland), in RPMI 1640
medium (Gibco), supplemented with 5% heat-inactivated fetal bovine
serum (Flow Laboratories, Irvine, Scotland) derived from a single batch,
and 2 HIML-glutamine (Gibco) at 36.5°Cin a humidified atmosphere
of 5% CO2 in air. Each cell line was used over a restricted number of
passages (maximum of 10), using stocks held in liquid nitrogen (see
Table 1). For subculturing, monolayers were detached using 0.01%
irypsin (Difco Laboratories, London, England; Difco 1:250) plus
0.003% versene (BDH Chemicals, Poole, England; EDTA disodium
salt) in PBSA.
PPLO Screening. PPLO contamination was screened by staining
with acetoorcein (5) and by inoculation of cells together with their
INTRODUCTION
culture medium into PPLO agar on a feeder layer of BHK21/13C cells,
as previously described (6).
The natural history and therapy of human tumors are dictated
Isozyme Analysis. A maximum of 13 polymorphic enzymes was
primarily by the site and histopathology of the disease. Never
isozyme typed in each cell line. Exponentially growing cells were
theless, among tumors of any one histológica! type there is a detached, washed once in medium and once in PBSA, centrifugea at
broad spectrum of biological behavior and response to particu
250 x g for 10 min between each step, transferred in PBSA in 1-ml
lar treatment regimen. Consequently model systems are needed
aliquots containing approximately 10 cells to freezing vials (Nunc),
which accurately represent not only individual tumors, but also and again centrifuged at 250 x g for 10 min. The PBSA was decanted,
reflect the range of properties of each histológica! type of and the cells were left to air dry for 15 min before storage in liquid
nitrogen until analysis. The enzymes were typed by horizontal starch
disease. One model system that might encompass these dual
gel electrophoresis as previously described (7), except for a-fucosidase
criteria is a panel of continuous cell lines derived from tumors
which was examined by isoelectric focusing (8).
of one histological type. In this study we investigated the extent
Tumorigenicity in Nude Mice. Exponentially growing cells were de
to which these two criteria are fulfilled by a panel of cell lines tached
and washed twice in serum and glutamine-free (unsupplemented)
derived from TCC2 of the human bladder. No previous attempt
medium. Approximately 10 cells in 0.1 ml of unsupplemented medium
has been made systematically to examine under standardized
were injected s.c. into the flank of a nude mouse (nu/nu), using at least
conditions the properties of a large series of human TCC cell four replicates for each cell line. The mice were examined weekly for
lines, although there are numerous reports concerning individ
gross evidence of tumor development and killed by cervical dislocation
ual or small numbers of lines ( 1-4).
when tumors had grown to 0.5-1.0 cm in diameter. The tumors were
excised, fixed in Baker's formol calcium, and processed using routine
histological procedures. Paraffin sections were stained with hematoxyMATERIALS AND METHODS
lin:eosin.
Cytology. Air-dried cell smears were stained with May-Grunwald/
Details concerning the origin of the lines used in this study are shown
Giemsa (R. A. Lamb, BDH Chemicals, London, England), and cell
in Table 1. Eighteen lines were derived from transitional cell carcino
smears fixed in 95% alcohol were Papanicolaou stained (Ortho Diag
mas, one from a squamous cell carcinoma, and three from normal
nostics, High Wycombe, England).
urothelium. With the exceptions of 253J, VM-CUB-I, and VM-CUBColony-forming Efficiency on Plastic. Exponentially growing cells
III (obtained from Dr. J. Fogh, Sloan-Kettering Institute for Cancer
were detached enzymatically, and single-cell suspensions were prepared
Research, Rye, NY), all the lines were obtained either from the origi
by repeated passage through a 19 gauge needle. Serial 50% dilutions
nator or laboratory of origin: RT4 and RT112 (this laboratory);
were plated in triplicate in 5-cm plastic Petri dishes (Nunc) to yield
viable cell numbers ranging from 50-6400/dish. The cells were incu
Received 7/2/85; revised 12/27/85; accepted 3/17/86.
1To whom requests for reprints should be addressed.
bated for between 8 and 28 days, depending on the growth rate of the
2The abbreviations used are: TCC, transitional cell carcinoma; ADA, adenocell line. Cell lines requiring an incubation period of 14 days or more
sine deaminase; CFE, colony-forming efficiency; LI, tritiated iIn mulino labeling
to develop colonies were medium-changed at 7-day intervals. The
index; PBSA, calcium- and magnesium-free phosphate-buffered saline; PDT,
population doubling time; PPLO, pleuropneumonia-like organism.
colonies were fixed in methanol (BDH Chemicals; Analar grade) and
3630
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1986 American Association for Cancer Research.
CHARACTERIZATION
OF HUMAN UROTHELIAL
CELL LINES
Table 1 Origin of cell lines
prior
r culture
of
stage
of tumor
Cell line
gradeG,G,NRG,G,G,G,G4G.G,G,G.NRNRG,G)NRY
established1970197219751975NR1972196719721972197319731974197519751975
patientFMMMMMMMMFFFMMMMMMNRMFPatient's
therapyNoneNRNRNoneNone4200
diseaseNR°T2
of
biopsyRecurrence
designationT24MGH-U1
nos.51-60103-112129-13854-6387-9634-4367-7682-9136-4535-4435-4459-6814-2316-2549-5831-40132-14178-8723-3211-2018-27Origin
bladderBladder
in
(EJ)MGH-U2HU456HU961TJ82COTRT4253JHT1197HT
primaryBladder
primaryBladder
primaryBladder
primaryBladder
primaryRecurrence
minimumNRTiT,T,TjT«T;
preop-erativelyGold
rads
yrearlierNRNoneNoneNoneNoneNRNRNone4000
grains 2
bladderRetroperitoneallymph
in
node metas
tasisRecurrence
bladderBladder
in
primaryBladder
1376RT112TCCSUPVM-CUB-IVM-CUB-IIIPS1COLO
primaryBladder
primaryBladder
primaryBladder
primaryBladder
primaryBladder
primaryBladder
232KK47SCaBERHCV29HS0767HU609Passage
minimumT2
minimumNRT4NRNRT,T»NRTjHistológica!
mopreoperativelyNRNoneNRNRRef.404142434344
rads 2
primanBladder
primarysquamous
cell car
cinomaNormal
frompatient
bladder
with blad
cancerNormal
der
frompatient
bladder
with pros
cancerNormal
tate
frompatient
ureter
renalcellwith
cancerClinical
" NR, not recorded.
* C. C. Rigby, personal communication.
stained with 10% Giemsa (Gurrs Improved R66; BDH Chemicals).
Colonies containing more than 50 cells were scored using a binocular
dissecting microscope, and the mean CFE derived from a minimum of
three separate experiments was calculated.
CFE =
no. of colonies
x 100
no. of viable cells seeded
Population Doubling Times. Exponentially growing cells were plated
in 3.5-cm dishes (Nunc) containing 5 ml of medium, at numbers ranging
in serial 50% dilutions from 768,000-24,000 cells/dish, with 2 repli
cates for each cell concentration at each time point. After 24, 48, 72,
and 96 h of incubation the cells were enzymically detached, and a single
cell suspension was produced by repeated passage through a 19 gauge
needle. Cells were suspended in a known volume of filtered PBSA, and
the numbers in two replicate 0.5-ml aliquots of each sample were
determined using a Coulter Counter (Coulter Electronics, Luton, Eng
land). The mean results from a minimum of two experiments were
plotted on semilogarithmic graph paper, and the PDT was calculated
for cell concentrations growing exponentially between 48 and 96 h after
initial plating, using the formula PDT = In2/ [In (Nt/No)]~', where / is
time interval (48 h), Nt is number of cells at 96 h, and No is number of
cells at 48 h.
Labeling Indices. Exponentially growing cells were plated in 5-cm
plastic Petri dishes containing 5 ml of medium. Following a 48-h
incubation period to facilitate the resumption of an exponential growth
rate, the medium was discarded, and fresh medium containing [methyl3H]thymidine (20 ¿iCi/ml;Amersham International, Amersham, Eng
land; 25 Ci/mmol) was added to each dish. After a 15-min pulse, the
dishes were rinsed 3 times with unsupplemented medium at 4°Cfor 5
min each, fixed by three 5-min washes in 95% ethanol at 4"C, and left
inverted to air dry. The fixed cells were coated with chrome alum/
gelatin (9) and prepared for autoradiography as described previously
(10). Briefly, the cells were covered with a layer of Ilford K5 emulsion
(Ilford Nuclear Research, Knutsford, England) diluted 1:3 with distilled
water. Following a 7-day exposure at 4°C,the emulsion was developed
using Kodak D19 (Kodak-Pathé,Chalon-Sur-Saone, France) diluted
1:1 with distilled water. A minimum of 500 nuclei was counted, and
those covered by more than 15 individual silver grains were scored as
positive. Data are derived from two separate experiments.
Time-lapse Cinemicroscopy. The apparatus used has been described
(11). Five-cm Petri dishes containing cells were transferred to an
Olympus IMT inverted microscope fitted with a Perspex incubator,
Bolex H16J camera, and Olympus control unit. Kodak 7454 or Kodak
Infocapture AHU 1454 microfilm was used, and the negatives were
analyzed on a LW analyzing projector.
RESULTS
Data from 22 cell lines are described. Five of the lines (PS1,
COLO232, J82, KK47, and J82 COT) were PPLO positive on
repeated examination and were examined in less detail in order
to avoid contamination.
Isozyme Analysis. Among the 22 cell lines, there were 16 that
differed at one or more alÃ-eles(Table 2), indicating each is
unique in origin. In contrast, T24 and five other lines (J82,
MGH-U1, MGH-U2, HU456, HU961T) were identical at each
locus, indicating that these almost certainly have been crosscontaminated. Original stocks of J82, designated J82 COT,
differ from T24 and have an isozyme pattern identical to that
of another cell line derived from the same patient, providing
further evidence that the subline J82 has been cross-contami
nated with T24 in some stocks. Two further lines, VM-CUB-I
and VM-CUB-III, had identical isozyme phenotypes, suggest
ing cross-contamination. None of the cell lines had the same
isozyme pattern as HeLa cells.
The three lines derived from normal urothelium (HCV29,
HS0767, HU609) all had an ADA isozyme of slow electrophoretic mobility (designated "tissue" in Table 2). This phenotype
is common in untransformed
cells in culture and, among the
3631
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1986 American Association for Cancer Research.
CHARACTERIZATION
OF HUMAN
UROTHELIAL
CELL LINES
Table 2 Isozyme profiles of the bladder cell lines
Cell line
designation
Enzyme loci
PGM1"
PGM3
GOTM
COTS
ESD
AK1
ADA
0RT4
2-1TCCSUP
2253J
1HTII97
1HT
2VM-CUB-I
1376
2-1VM-CUB-I11
2-1KK47
1COL0232
2-1PS1
1SCaBER
2-1HCV29
2-1HU609
1HSO767
PGD
G6PD
GLO
FUCA
PGP
ACPI
;ABI:ABI:ABI:ABI:ABI;A
B
i
T24MGH-UI
(EJ)MGH-U2HU456HU961TJ82J82COTRT1I2
:A
:2-1
2-1MD
]2I2-112-1
2-1'issue
2)
2-1A
A
1A
A
B
B
B
B
ND
B
2
J-1.-1
BABABABMD
B
B
B
A
1MD
B
2
BMD
B
ND
11!-l
BMD
1fissue
A
2-1
BMD
1MDMDBABABABABABACABNDABABA-1
;[issiliA
ND
1
A1
IPissue
A
B
2
1ND
CAVD
2-122
1.-1MD11MDMDMD
1VDMDMDMD
:.-i.-i:!-l:!-l:!-ll!!M!-l'A
AC
B
ND
1;_;.¡_)_;_!-l!-l_
B
* PGM l and 3, first and third loci of phosphoglucomutase; GOTM and S, mitochondria! and soluble glutamate-oxaloacetate transaminase; ESD, esterase D; AKI.
adcnylatc kinase; PGD, 6-phosphogluconate dehydrogenase; G6PD, glucose-6-phosphate dehydrogenase; GLO, glyoxalase: FUCA, a-fucosidase; PGP, phosphoglycollaie phosphatase; ACPI, first locus of acid phosphatase; ND. not done: Tissue, not typable because ADA complexed with glycoprotein.
2A
ND
1A
1A
2A
:A
1A
tumor-derived lines, was only observed in HT1197.
Tumorigenicity. Details concerning the growth of the cell
lines as xenografts are summarized in Table 3. Two tumorderived lines (T24 and TCCSUP) and the three lines derived
from normal urothelium (HCV29, HU609, HS0767) failed to
develop tumors during a maximum follow-up period of 2 yr.
Independent morphological descriptions of each tumor by
two experienced urological histopathologists were in agreement
(summarized in Table 3). Replicate xenografts derived from the
same cell line had similar morphologies. The tumors produced
by RT4 and RT112 were morphologically similar to those of
the original biopsies taken in 1967 and 1973, respectively (Figs.
1 to 4). Areas of squamous metaplasia were more common in
the xenografts, perhaps reflecting differences in the hormonal
environment in the mice. HT 1376 and HT 1197 produced
poorly differentiated tumors similar in microscopic appearance
to those illustrated in the original description of these lines
(12). Thus, in the cases in which direct comparison could be
tumorigenicityCell
line
designationT24MGH-UI
made of the xenograft and the tumor of origin, the light
microscopic appearances were similar.
The cross-contaminated sublines of T24 ranged from being
highly tumorigenic with all implants developing large tumors
rapidly (MGH-UI and MGH-U2), through intermediate de
grees of tumorigenicity (HU961T and HU456), to failure to
develop tumors under these conditions (T24). Furthermore, the
morphologies of the tumors produced by the sublines differed.
T24 cells from another laboratory produced a xenograft with a
spindle-cell morphology, and this component was also seen in
the xenografts derived from MGH-UI and MGH-U2. HU456
and HU961T produced transitional cell carcinomas with areas
of squamous metaplasia.
The cytological appearances of the cell lines in vitro were all
consistent with an origin from transitional cell carcinomas. The
squamous metaplasia observed following xenotransplantation
was not apparent in vitro, although there was considerable
variation in the degree of cellular differentiation.
Table 3 Cell line morphology and
appear
cellsDifferentiated
ance of cultured
gradeDid
histopathology and
tumorsGj,
not form
TCCDifferentiated
MR*Gj,
possibly TCC with spindle cell areas and high
(EJ)MGH-U2HU456HU961TRT112RT4253JHTI376HT1197VM-CUB-11ITCCSUPCytological
TCCDifferentiated
MR<possibly TCC with spindle cell areas and high
TCCDifferentiated
metaplasia(
. , TCC with squamous
TCCDifferentiated
metaplasiaGi.2,
' •
<.TCC with squamous
TCCDifferentiated
glandularmetaplasiaGì,
TCC with focal squamous and
TCCDifferentiated
mo)"
TCCTCC,
probably dif
ferentiatedTCC,
mostly differ
entiatedPleomorphic
TCC,mixture
differen
tiated
anaplas-tic
and
squa-niiiiil
cells with
changeTCC,
probably dif
ferentiatedConsistent
withdifferentiated
TCCXenograft
(time
taken for tumors to
grow
mice)0/8
in nude
mo)4/4(1
(after 24
mo)4/4(1
mo)3/8
mo)3/3
(6-20
mo)3/3(1
(5-6
mo)4/4
MRGi_2,
TCC with squamous metaplasia and high
metaplasiaGj,
TCC with glandular
mo)2/2
(2-5
mo)3/3
(6- 12
extensivesquamous
squamous cell or TCC with
MRGÌ, metaplasia and high
carcinomaGj,
squamous cell
mo)4/4
(1
carcinomaDid
squamoid cell
mo)0/8
(2
not form tumorsTumorigenicity
mo)4/4
(1-20
(after 24
MR, mitotic rate.
3632
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1986 American Association for Cancer Research.
CHARACTERIZATION
OF HUMAN UROTHELIAL
Fig. 1. Parent tumor of RT112. Papillary transitional cell carcinoma of the
human bladder. H & E, x 240.
CELL LINES
F'8- 3- Parent tumor of RT4- Papillary transitional cell carcinoma of the
h""""1 bladder. H & E, x 240.
i*.l::v»-?;^'Ã-V:r^:*•;...-.;.,¿. ?
5; ^,-f;
88ÄIP
ÃŒli;
Fig. 2. RT1I2 xenograft showing similar morphology to that of the parent
tumor in Fig. 1. H & E, x 240.
r ;-'. „.-•v*v»>»v
.
IfÜä
Fig. 4. RT4 xenograft. showing similar morphology to that of the parent
tumor in Fig. 3. H & E, x 240.
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1986 American Association for Cancer Research.
CHARACTERIZATION
OF HUMAN UROTHELIAL
CELL LINES
Growth Characteristics. The in vitro growth characteristics of
the cell lines are listed in Table 4. The similarities in these
features between T24 and its cross-contaminated sublines con
trast with the wide variation among the other distinct lines.
There is some association between a long PDT and both low
CFE and LI. An exception is HT1197 which, despite having
the longest PDT and lowest CFE, has a relatively high LI of
37.3%. This observation was investigated further using timelapse cinemicroscopy to compare the cell division times and
cell loss rates of HT1197 and T24 (see Table 5). The lack of
association between the PDT and LI of HT1197 may be ex
plained by the high death rate of HT1197 (35%) compared with
that of T24 (0.5%).
Xenotransplantation has been used to demonstrate that most
human tumor cell lines retain a neoplastic phenotype and
produce xenografts with a morphology compatible with that of
the tumor of origin (21, 22). In the largest series of cell lines
studied, 127 of 162 (78%) produced tumors, and in every case
the histopathology correlated with that of the tumor of origin
(23). In this and earlier studies of both human (2, 3) and rat
(24) bladder tumor cell lines, the ability to form tumors with
morphologies similar to those of the tumors of origin was
retained.
Tumorigenicity was highly variable among the five T24 sublines tested. Cloning of one of these sublines, MGH-U1 (des
ignated EJ in some studies), also resulted in lines differing
widely in tumorigenicity (25). Loss of tumorigenicity has also
been observed in melanoma (26) and other cell lines (23). These
DISCUSSION
differences may reflect changes in the immunogenicity of the
cells, perhaps reflected by the changes we observed in the T24
This paper describes certain biological characteristics of a sublines in cell surface antigen expression demonstrated with
panel of cell lines derived from TCC of the human bladder,
monoclonal antibodies (14). T24 and its sublines could provide
maintained under identical and standardized culture conditions.
a model system for studying certain of the factors involved in
In parallel studies, we measured in vitro drug sensitivities (13) tumorigenicity, and they are of particular interest as T24 was
and reactivities with a panel of monoclonal antibodies derived
the first human cell line shown to contain a transfectable
from one of these cell lines (14). These data provide a basis on oncogene (27).
which to evaluate a panel of cell lines as a model system for
The in vitro growth characteristics of the T24 sublines were
tumors of this histológica! type.
similar, contrasting with the wide variation among the distinct
Isozyme analysis can be used to identify cross-contamination
cell lines. The relative stability and reproducibility of these data
between cell lines of the same or another species and, if tissue
probably result from the use of standardized conditions. All the
from the patient is available, establish that the cells are derived
lines were grown in one type of tissue culture medium, supple
from that individual (15). Isozyme phenotypes are retained in mented with a single batch of fetal bovine serum over a re
vitro and usually remain stable over many years in culture (16). stricted passage range. Alteration in any one of these conditions
In this study, 16 lines were shown to have distinct isozyme
can radically alter the properties of cells in vitro, and it is
phenotypes, indicating derivation from different individuals,
essential that comparisons between cell lines are made using
while 6 lines appeared to be cross-contaminated. Bladder tumor
standardized culture conditions. The growth rates of all of these
cell lines appear to have been cross-contaminated with T24
lines are considerably higher than those observed in human
cells in at least 3 separate laboratories. The isozyme patterns
bladder
tumors, either as measured directly in patients (28) or
of some of these lines have been described previously (15, 17indirectly in biopsies in vitro (29). Similarly, the colony-forming
20), and our data are in agreement.
efficiencies on plastic (ranging from 3-87%) were far higher
than those achieved by cells cultured in agar directly from
Table 4 Growth characteristics of cell lines
human bladder tumor biopsies (30).
In addition to characterizing cell lines derived from TCC,
Cell line
Population douefficiency on
(h)MGH-U2MGH-U1
designation
bling time
plastic
(%)877657618416222752743Labeling
index535254515129423927321237
preliminary studies were made on three lines derived from
normal urothelium. In support of their origin from normal
(EJ)HU961TT24HU456RT1I2VM-CUB-3TCCSUP253JHT1376RT4HT1197192021212124262828313761Colony-forming
tissue, all three lines expressed the untransformed phenotype
of the ADA enzyme, in contrast to only 1 of 13 of the tumorderived lines. The untransformed phenotype is seen in virtually
all normal adult fibroblast cultures, but it is rare in transformed
cells in culture (31). However, the ability of cells to grow
indefinitely in vitro is usually regarded as an abnormal feature,
and therefore the relevance of these lines as a model for normal
human urothelium is uncertain. Normal urothelium can be
cultured directly from rat (32, 33) and human (34) bladders,
Table 5 Estimates of cell loss rate and intermitotic times ofHT1197 and T24 lines followed for two cell generations by time-lapse cinemicroscopy
Some cells were lost from the field of view, as tabulated, either as a result of migration or detachment from the substrate.
15812211827.8-63.54S.6HT1197Generation
2397141323.6-102.248.6Total971935
I77207513.4-26.417.1T24Generation
215216113512.3-20.916.4Total229181
Cell
lossTotal
cellsLost
viewDiedIntermitotic
from field of
(36%)3123.6-102.246.9Generation
(0.5%)21012.3-26.416.6
(h)Total time
cellsRangeMeanGeneration
3634
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1986 American Association for Cancer Research.
CHARACTERIZATION
OF HUMAN UROTHELIAL
and it is now possible to propagate large numbers of human
normal urothelial cells in defined medium (35). Spontaneous
transformation in such cultures has been observed with rat, but
not human, urothelium (36).
The ultimate goal of these studies is to assess the value of
these cell lines as a model for TCC. Cell lines can be selected
which are representative of their tumors of origin on the basis
of isozyme analysis and xenotransplantation.
Nevertheless,
some characteristics, such as growth rate and clonogenicity,
alter as a result of in vitro culture, and these changes must be
taken into account when using cell lines as a model system.
The panel of cell lines shows a wide range of morphology,
degree of histological differentiation, biochemical properties,
growth characteristics, sensitivities to chemotherapeutic drugs
(13), and reactivities with monoclonal antibodies (14). Similar
heterogeneity has been observed in cell lines derived from
human lung tumors (37), ovarian carcinomas (38), and mela
nomas (26). These data indicate that a panel of cell lines could
be selected reflecting the spectrum of properties exhibited by
each histological type of tumor. For use as a model system, it
is also essential that the properties of the cell lines remain
relatively stable in long-term culture. However, tumors are
inherently heterogeneous and capable of generating diversity,
for instance by developing drug-resistant phenotypes (39). Sta
bility in long-term culture was investigated using the T24 sublines, which had been maintained in various laboratories under
different culture conditions, separately for periods of up to 10
yr. The sublines were similar in their isozyme patterns, growth
rates, and in vitro drug sensitivities, but markedly different in
tumorigenicity and reactivity with certain monoclonal antibod
ies (14). To limit such differences and ensure comparability
between and within laboratories, it will be necessary to use a
common stock of each cell line over a limited number of
passages and under identical culture conditions.
In conclusion, these data support the concept that a panel of
cell lines derived from human TCC could provide a model
system for this disease, but also illustrate some of the inherent
problems. Disadvantages include the need for rigorous stand
ardization of culture conditions, the requirement to characterize
large numbers of cell lines and exclude cross-contamination
and PPLO infection, and the fact that certain characteristics
are not stable in long-term culture. Advantages include the
unlimited supply of cells derived from each human tumor and
the stability of features such as isozyme phenotypes, in vitro
drug sensitivities, and the ability to generate tumors histopathologically similar to the tumor of origin.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
ACKNOWLEDGMENTS
K. A. Wallace and D. E. Bennett carried out the xenografting
procedure in the Animal House Unit at the Imperial Cancer Research
Fund Laboratories. We are indebted to Dr. M. C. Parkinson and Dr.
K. M. Cameron for their support and advice.
28.
29.
30.
REFERENCES
31.
1. Hepburn, P. J., and Masters, J. R. W. The biological characteristics of
continuous cell lines derived from human bladder. In: G. T. Bryan and S. M.
Cohen (eds.). The Pathology of Bladder Cancer. Vol. 2, pp. 213-227. Boca
Raton, FL: CRC Press, Inc., 1983.
2. Grossman, H. B., Wedemeyer, G.. and Ren, L. UM-UC-1 and UM-UC-2:
characterization of two new human transitional cell carcinoma lines. J. I ml..
132: 834-837, 1984.
3. Kyriazis, A. A., Kyriazis. A. P.. McCombs, W. B.. and Peterson, W. D.
Morphological, biological, and biochemical characteristics of human bladder
transitional cell carcinomas grown in tissue culture and in nude mice. Cancer
Res.. 44:3997-4005, 1984.
32.
33.
34.
35.
3635
CELL LINES
Lin, C-W., Lin, J. C., and Prout, G. R. Establishment and characterization
of four human bladder tumor cell lines and sublines with different degrees of
malignancy. Cancer Res.. 45: 5070-5079, 1985.
Fogh, J., and Fogh, H. A method for direct demonstration of pleuropneumonia-like organisms in cultured cells. Proc. Soc. Exp. Biol. Med., 117:899901, 1964.
Dickson, C., Elkington, J., Hales, A., and Weiss, R. Antibody-mediated
neutralization of virus is abrogated by Mycoplasma. Infect. Immun., 28:649653, 1980.
Harris, H., and Hopkinson, D. A. Handbook of Enzyme Electrophoresis in
Human Genetics. Amsterdam: North Holland Publishing Company, 1976.
Turner, B. M., Turner, V. S., Beratis, N. G., and Hirschhorn, K. Polymor
phism of human alpha-fucosidase. Am. J. Hum. Genet., 27: 651-661, 1975.
Brooks, R. F. The kinetics of serum-induced initiation of DNA synthesis in
BHK21/C13 cells, and the influence of exogenous adenosine. J. Cell Physiol.,
«6:369-378,1975.
Brooks. R. F., Riddle, P. N., Richmond, R. N., and Marsden, J. The GI
distribution of "Gl-less" V79 Chinese hamster cells. Exp. Cell Res., 148:
127-142, 1983.
Riddle, R. N. Time-lapse Cinemicroscopy. London: Academic Press, 1979.
Rasheed, S., Gardner, M. B., Rongey, R. W., Nelson-Rees, W. A., and
Arnstein, P. Human bladder carcinoma: characterization of two new tumor
cell lines and search for tumor viruses. J. Nati. Cancer Inst.. 58: 881-890,
1977.
Masters, J. R. W., Hepburn, P. J., English, P., and Oliver, R. T. D. Spectrum
of in vitro drug sensitivities of human bladder cancer continuous cell lines.
Proc. Am. Assoc. Cancer Res., 28: 328, 1984.
Trejdosiewicz, L. K., Southgate, J., Donald, J. A., Masters, J. R. W.,
Hepburn, P. J., and Hodges, G. M. Monoclonal antibodies to human uro
thelial cell lines and hybrids: production and characterization. J. Urol.. 133:
533-538, 1985.
Povey, S., Hopkinson, D. A.. Harris. H., and Franks, L. M. Characterization
of human cell lines and differentiation from HeLa by enzyme typing. Nature
(Lond.), 264:60-63, 1976.
Wright, W. C., Daniels, W. P., and Fogh, J. Stability of polymorphic enzyme
phenotypes in human tumor cell lines. Proc. Soc. Exp. Biol. Med., 162:503509, 1979.
Wright, W. C., Daniels, W. P., and Fogh, J. Distinction of seventy-one
cultured human tumor cell lines by polymorphic enzyme analysis. J. Nati.
Cancer Inst., 66: 239-247, 1981.
Dracopoli, N. C., and Fogh, J. Loss of heterozygosity in cultured human
tumor cell lines. J. Nati. Cancer Inst., 70: 83-87, 1983.
Dracopoli, N. C., and Fogh, J. Polymorphic enzyme analysis of cultured
human tumor cell lines. J. Nati. Cancer Inst., 70:469-476, 1983.
O'Toole, C. M., Povey, S., Hepburn, P., and Franks, L. M. Identity of some
human bladder cancer cell lines. Nature (Lond.), 301:429-430, 1983.
Povlsen, C. O., Spang-Thomsen, M., Rygaard, J., and Visfeldt, J. Heterotransplantation of human malignant tumours to athymic nude mice. In: S.
Sparrow (ed.), Immunodeficient Animals for Cancer Research, pp. 95-103.
London: Macmillan Press, 1980.
Sharkey, F. E.. and Fogh. J. Considerations in the use of nude mice for
cancer research. Cancer Metastasis Rev., 3: 341-360, 1984.
Fogh, J.. Fogh. J. M.. and Orfeo, T. One hundred and twenty-seven cultured
human tumor cell lines producing tumors in nude mice. J. Nati. Cancer Inst..
59:221-225. 1977.
Chlapowski, F. J., Minsky. B. D.. Jacobs. J. B., and Cohen, S. M. Effect of
a collagen substrate on the growth and development of normal and tumorigenie rat urothelial cells. J. Urol., 130: 1211-1216, 1983.
Hastings, R. J.. and Franks, L. M. Cellular heterogeneity in a tissue culture
cell line derived from a human bladder carcinoma. Br. J. Cancer, 47: 233244, 1983.
Tveit, K. M.. and Pihl, A. Do cell lines in vitro reflect the properties of the
tumours of origin? A study of lines derived from human melanoma xenografts. Br. J. Cancer, 44: 775-786, 1981.
Shih, C., Padhy, L. C., Murray, M., and Weinberg, R. A. Transforming genes
of carcinomas and neuroblastomas introduced into mouse fibroblasts. Nature
(Lond.), 290: 261-264, 1981.
Shackney, S. E., McCormack, G. W., and Cuchural, G. J. Growth rate
patterns of solid tumors and their relation to responsiveness to therapy. Ann.
Intern. Med., 89: 107-121. 1978.
Levi, P. E., Cooper, E. H., Anderson, C. K., and Williams, R. E. Analyses
of DNA content, nuclear size, and cell proliferation of transitional cell
carcinoma in man. Cancer (Phila.), 23:1074-1085, 1969.
Sarosdy, M. F., Lamm, D. L.. Radwin. H. M., and Von Hoff, D. D.
Clonogenic assay and in vitro chemosensitivity testing of human urologie
malignancies. Cancer (Phila.), 50: 1332-1338, 1982.
Inwood, M., Povey, S., and Delhanty, J. D. A. Comparison of isozymes in
fetal, adult and transformed fibroblasts. Cell. Biol. Int. Rep., 4: 327-335,
1980.
Pauli, B. U., Anderson, S. N.. Memoli, V. A., and Kuettner, K. E. The
isolation and characterization in vitro of normal epithelial cells, endothelial
cells and fibroblasts from rat urinary bladder. Tissue Cell, 12:419-435,1980.
Chlapowski, F. J., and Haynes, L. The growth and differentiation of transi
tional epithelium in vitro. J. Cell Biol., 83: 605-614, 1979.
Reznikoff, C. A., Johnson, M. D., Norback, D. H., and Bryan, G. T. Growth
and characterization of normal human urothelium in vitro. In Vitro (Rockville), 19: 326-343, 1983.
Kirk, D., Kagawa, S., Vener, G., Shankar Narayan, K., Ohnuki, Y.. and
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1986 American Association for Cancer Research.
CHARACTERIZATION
36.
37.
38.
39.
40.
41.
42.
43.
OF HUMAN UROTHELIAL
Jones, L. W. Selective growth of normal adult human urothelial cells in
serum-free medium. In Vitro (Rockville), 21: 165-171, 1985.
Johnson, M. D., Bryan, G. T., and Reznikoff, C. A. Serial cultivation of
normal rat bladder epithelial cells in vitro. J. Uro!.. 33:1076-1081, 1985.
Baylin, S. B., Jackson, R. H., Lennarz, W., and Shaper, J. H. The spectrum
of human lung cancer cells in culture: a potential model for studying molec
ular determinants of tumor progression and metastasis. In: G. L. Nicholson
(ed.), Cancer Invasion and Metastasis: Biologic and Therapeutic Aspects, pp.
281-291. New York: Raven Press, 1984.
Hamilton, T. C., Young, R. C., and Ozols, R. F. Experimental model system
of ovarian cancer: applications to the design and evaluation of new treatment
approaches. Semin. Oncol., //: 285-298, 1984.
Fogh, J., Dracopoli, N., Loveless, J. D., and Fogh, H. Cultured human tumor
cells for cancer research: assessment of variation and stability of cultural
characteristics. Prog. Clin. Biol. Res., 89: 191-223, 1982.
Bubenik, J., Baresova, M., Viklicky, V., Jakoubkova, J., Sainerova, H., and
Donner, J. Established cell line of urinary bladder carcinoma (T24) contain
ing tumour-specific antigen. Int. J. Cancer, //: 765-773, 1973.
Evans, D. R., Irwin, R. J., Havre, P. A., Bouchard. J. G.. Kato, T., and
Prout, G. R. The activity of the pyrimidine biosynthetic pathway in MGHUl transitional carcinoma cells grown in tissue culture. J. Urol., 117: 712719, 1977.
Daly. J. J., Prout, G. R., Hagen, K., Lin, J. C, Kamali, H. M., Plotkin, G.
M., and Wolf, G. Identification of malignant transitional cells by their
binding of fluorescein-labeled lectin. Surg. Forum, 30: 565-567, 1979.
Vilien, M., Christensen, B., Wolf, H., Rasmussen, F., Hou-Jensen, C, and
Povlsen, C. O. Comparative studies of normal, "spontaneously" transformed
and malignant human urothelium cells in vitro. Eur. J. Cancer Clin. Oncol.,
79:775-789, 1983.
44. O'Toole, C., Price, Z. H., Ohnuki, Y., and Unsgaard, B. Ultrastructure,
karyology and immunology of a cell line originated from a human transitional
cell carcinoma. Br. J. Cancer, 38:64-76, 1978.
CELL LINES
45. Rigby, C. C., and Franks, L. M. A human tissue culture cell line from a
transitional cell tumour of the urinary bladder: growth, chromosome pattern
and ultrastructure. Br. J. Cancer, 24: 746-754, 1970.
46. Elliott, A. Y., Cleveland, P., Cervenka, J., Castro, A. E., Stein, N., Hakala,
T. R., and Fraley, E. E. Characterization of a cell line from human transi
tional cell cancer of the urinary tract. J. Nati. Cancer Inst., 53: 1341-1349,
1974.
47. Nayak, S. K., O'Toole, C., and Price, Z. H. A cell line from an anaplastic
transitional cell carcinoma of human urinary bladder. Br. J. Cancer, 35:142151, 1977.
48. Williams, R. D. Human urologie cancer cell lines. Invest. Urol., 17: 359363, 1980.
49. Sanford, E. J., Geder, L., Dagen, J. E., Laychock, A. M., Ladda, R., and
Rohner, T. J. Establishment and characterization of a new human urinary
bladder carcinoma cell line (PS-1). Invest. Urol., 16:246-252, 1978.
50. Moore, G. E., Morgan, R. T., Quinn, L. A., and Woods, L. K. A transitional
cell carcinoma cell line. In Vitro (Rockville), 14: 301-306, 1978.
51. Hisazumi, H., Kanokogi, M., Nakajima, K., Koboyashi, T., Tsukahara, K.,
Naito, K., and Kuroda, K. Established cell line of urinary bladder carcinoma
(KK-47): growth, heterotransplantation, microscopic structure and chromo
some pattern. Jpn J. Urol., 70:485-494, 1979.
52. O'Toole, C., Nayak, S., Price, Z., Gilbert, W. H., and Waisman, J. A cell
line (SCaBER) derived from squamous cell carcinoma of the human urinary
bladder. Int. J. Cancer, 17: 707-714, 1976.
53. Bean, M. A., Pees, H., Fogh, J. E., Grabstald, H., and Oettgen, H. F.
Cytotoxicity of lymphocytes from patients with cancer of the urinary bladder:
detection by a 3H-proline microcytotoxicity test. Int. J. Cancer, 14:186-197,
1974.
54. Smith, H. S., Springer, E. L., and Hackett, A. J. Nuclear ultrastructure of
epithelial cell lines derived from human carcinomas and nonmalignant tis
sues. Cancer Res., 39: 332-344, 1979.
55. Kieler, J. F. Invasiveness of transformed bladder epithelial cells. Cancer
Metastasis Rev., 3: 265-296, 1984.
3636
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1986 American Association for Cancer Research.
Tissue Culture Model of Transitional Cell Carcinoma:
Characterization of Twenty-two Human Urothelial Cell Lines
John R. W. Masters, Peter J. Hepburn, Lawrence Walker, et al.
Cancer Res 1986;46:3630-3636.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/46/7/3630
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1986 American Association for Cancer Research.