[CANCER RESEARCH 61, 6281– 6289, August 15, 2001]
Implication of Tyrosine Kinase Receptor and Steel Factor in Cell Density-dependent
Growth in Cervical Cancers and Leukemias1
Julio R. Caceres-Cortes,2 Jose A. Alvarado-Moreno, Kazuo Waga, Rosalva Rangel-Corona, Alberto Monroy-Garcia,
Leticia Rocha-Zavaleta, Julia Urdiales-Ramos, Benny Weiss-Steider, Andre Haman, Patrice Hugo, Roland Brousseau,
Trang Hoang
Laboratories of Oncology [J. R. C-C., J. A. A-M., R. R-C., J. U-R.] and Immunobiology, [A. M-G.], Research Unit in Cell Differentiation and Cancer, Facultad de Estudios
Superiores Zaragoza [B. W-S.], and Molecular Biology Department, Biomedical Research Institute [L. R-Z.] Universidad Autonoma de Mexico, Mexico 15000; Institut de
Recherches en Biotechnologie, Montreal, H4P 2R2 Quebec, Canada [R. B.]; Procrea, Montreal, H4P 2R2, Quebec, Canada [P. H.]; and Laboratory of Hemopoiesis and
Leukemia, Clinical Research Institute of Montreal, Montreal, H2W 1R7, Quebec, Canada [K. W., A. H., T. H.]
ABSTRACT
Cell-cell interaction is important in the expansion of leukemic cells and
of solid tumors. Steel factor (SF) or Kit ligand is produced as a membranebound form (mSF) and a soluble form. Because both primary gynecological tumors and primary leukemic cells from patients with acute myeloblastic leukemia (AML) have been shown to coexpress c-Kit and SF, we
addressed the question of whether mSF could contribute to cell interaction
in these cancers. Investigations on primary cervical carcinomas have been
hindered by the fact that the cells do not grow in culture. We report herein
the establishment of two cervical carcinoma cell lines, CALO and INBL,
that reproduce the pattern of SF/c-Kit expression observed in primary
tumor samples. In addition, these cells exhibit marked density-dependent
growth much in the same way as AML blasts. Using an antisense strategy
with phosphorothioate-modified oligonucleotides that specifically target
SF without affecting other surface markers, we provide direct evidence for
a role of mSF and c-Kit in cell interaction and cell survival in these
gynecological tumor cell lines as well as in primary AML blasts. Finally,
our study defines the importance of juxtacrine stimulation, which may be
as important, if not more, than autocrine stimulation in cancers.
INTRODUCTION
Cell-cell interaction is important in the expansion of leukemic cells
and solid tumors. Ligand-receptor pairs are frequently coexpressed in
human cancer cells, and the autocrine paracrine loops causing selfstimulation are thought to be involved in carcinogenesis (1–3). Human
solid tumor cell lines have been shown to produce hemopoietic
growth factors and display their receptors (4), suggesting that soluble
or membrane-bound growth factors may contribute to cell interaction.
Mutations at the W3 locus in mice have deleterious effects on germ
cells, melanocytes, and hematopoietic stem cells (reviewed in Ref. 5).
The W locus encodes the c-Kit receptor tyrosine kinase of which the
ligand is the product of the Steel (SI) locus. SF is produced as a
soluble form or a membrane-bound form (6, 7). There is considerable
evidence to suggest that c-Kit and SF are involved in the pathogenesis
of both hematological and nonhematological malignancies (8). For
Received 8/28/00; accepted 6/19/01.
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.
1
Supported by funds from the Council for National Research (to J. R. C-C.; Consejo
Nacional de Ciencia y Tecnologia No. 27880 M) and the Canadian Institute for Health
Research (to T. H.). J. R. C-C. is supported by a scholarship from the Council for National
Research.
2
To whom requests for reprints should be addressed, at Research Unit in Cell
Differentiation and Cancer, FES-Zaragoza, Universidad Autonoma de Mexico, J.C. Bonilla 66 Col. Ejercito de Oriente, Apdo Postal 9-020, Mexico D.F. CP 15000. Phone and
Fax: (525) 773-4108. E-mail: [email protected].
3
The abbreviations used are: W, white-spotting; IMDM, Iscove’s modified Dulbecco’s medium; SF, Steel factor; mSF, membrane Steel factor or Kit ligand; FAB, FrenchAmerican-British classification; PE, phycoerythrin; GM-CSF, granulocyte macrophage
colony-stimulating factor; TBS, Tris-buffered saline; TTBS, TBS with 0.2% Tween 20;
AML, acute myeloblastic leukemia; mAb, monoclonal antibody; HPV, human papillomavirus; IL, interleukin; IL-1R, IL-1 receptor; TdT, terminal deoxynucleotidyltransferase;
TUNEL, terminal deoxynucleotidyl transferase (Tdt)-mediated nick end labeling; AS-SF,
antisense Steel factor; PAKL, control oligonucleotide in which the sequence was shuffled.
example, the murine hemopoietic cell line 32D is nonleukemogenic.
Ectopic c-Kit expression confers to these cells a lethal leukemogenic
potential in vivo without an obvious effect on cell proliferation in vitro
(9). In addition, a very high percentage of primary AML blasts
express c-Kit (10), and the majority of fresh leukemic blasts are
growth stimulated by SF in vitro (11, 12). We and others have shown
that the growth of AML blasts is density-dependent and that SF (Kit
ligand) is the only cytokine that can replace cell interaction in AML
culture (9, 12, 13). Together, these results suggest that cell interaction
in AML may be mediated by SF/c-Kit interaction.
Molecular approaches have led to the concept that carcinomas arise
from the accumulation of a series of genetic alterations involving the
activation of proto-oncogenes and inactivation of tumor suppressor
genes. Despite the evidence that HPVs are involved in uterine cervix
carcinomas, the E6E7 genes of HPV16 alone cannot fully transform
human and rodent primary cells in vitro or cause tumor development
in immunodeficient animals. Full transformation requires cooperation
of E6E7 with other activated oncogenes (14 –16). It has been shown
that the oncogenes myc and ras are activated and that the tumor
suppressor genes p53 and Rb are altered, consistent with the concept
of multistep carcinogenesis (17). Whereas these molecular events may
confer a growth advantage to transformed cells, the mechanisms that
underlie the process of cell interaction in cancers remains to be
documented.
Transgenic mouse lines expressing HPV16 E6E7 oncogenes developed normally, but male mice exhibited a very high incidence of
Leydig cell tumors at 8 –10 months (18). The c-Kit receptor tyrosine
kinase and its ligand SF were coexpressed in all of the tumors
analyzed, and this coexpression of c-Kit/SF was also found in two
other Leydig cell tumor lines. Moreover, the proliferation of transgenic tumor cells was attenuated by treatment with a c-Kit-neutralizing antibody in vitro, strongly suggesting that tumorigenesis is closely
related to ligand-dependent receptor activation. A role for c-Kit in
tumorigenesis came from the demonstration that tumor formation
induced by the E6E7 transgene is attenuated in a Sl or W background
(19). These results indicated that c-Kit activity is an important determinant of testicular tumor development, although the function of
SF/c-Kit in the biology of HPV-induced gynecological tumors remains to be determined.
The incidence of cervical carcinoma in women in Peru is 54/
100,000, which is the highest in the world (20). Similarly, ⬃30% of
all of the malignant tumors in women in Mexico are uterine cervix
carcinomas (21). Invasive cervical cancer occurs most often in women
⬎40 years of age, and it is one of the main causes of cancer-related
death in Latin America. Current treatment includes surgery and radiotherapy or chemotherapy. Radiations and chemotherapeutic agents
induce apoptosis in cancer cells, but selectivity remains a key issue.
Drugs that target nucleic acids through Watson-Crick base paring
should provide unique specificities. Among those, antisense oligonucleotides designed against a specific RNA have been modified for
improved stability. The most promising are the phosphorothioates,
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DNA analogues in which the oxygen atoms are replaced by sulfur
atoms.
Both gynecological tumors (22) and colon tumor cell lines (23)
have been shown to coexpress c-Kit and SF. It has been reported that
autocrine/paracrine stimulation through this system may contribute to
tumorigenesis in lung, breast, and testicular malignancies (24 –28).
Much less is known about cervical carcinomas because of limited
availability of appropriate cellular models (29 –31). We report herein
the establishment of two carcinoma cell lines that reproduce the
pattern of SF/c-Kit expression observed in primary tumor samples.
Using an antisense strategy with phosphorothioate modified oligonucleotides directed against SF, we provide direct evidence for a role of
mSF and c-Kit in cell interaction and cell survival in these gynecological tumor cell lines, as well as in primary AML blasts.
MATERIALS AND METHODS
Source of Cells and Growth Factors. Peripheral blood from AML patients (Hotel-Dieu Hospital, Montreal, Canada) were obtained by informed
consent. AML blasts (Table 1) were isolated by centrifugation of peripheral
blood cells on a Ficoll-Hypaque gradient (Pharmacia Fine Chemicals, Uppsala,
Sweden). The cells were cryopreserved in FCS (Life Technologies, Inc., Grand
Island, NY) containing 10% DMSO and stored in liquid nitrogen until use. On
thawing, cells were ⬎90% viable.
The human cell line TF-1, a kind gift of Dr. T. Kitamura (DNAX, Palo Alto,
CA), was passaged 3 times weekly at 1.5 ⫻ 105 cell/ml in IMDM/10% FCS
containing human GM-CSF at 5 ng/ml as described previously (32).
The mouse 3T3 cell line p220 expressing membrane-bound human SF
(mSF) was a kind gift of Dr. D. Williams (Howard Hughes Medical Institute,
Indianapolis, IN) (33). Purified recombinant human GM-CSF was generously
provided by Dr. S. C. Clark (Genetics Institute, Cambridge, MA), and soluble
recombinant human SF was produced through transient transfection in SV40
transformed African green monkey kidney cells.
Cell Surface Analysis. For fluorescence-activated cell sorter analysis, cells
were labeled with a monoclonal mouse antihuman SF (7H6, IgG2a; kindly
provided by Dr. Keith Langley, Amgen, Thousand Oaks, CA), mouse antihuman c-Kit (1:10; Caltag, San Francisco, CA), or mouse antihuman HER-2
(Santa Cruz Biotechnology, Santa Cruz, CA; 1 ␮g/106 cells) followed by a
sheep antimouse coupled to PE at a final dilution of 1:40. Nonspecific binding
was blocked with a mixture of human immunoglobulin (10 ␮g/ml) in PBS
Table 1 List of patient samples
Aa
Patient sample
AML
AML
AML
AML
AML
AML
AML
32
34
45
53
55
58
72
FABb
%
blastsc
mSF Mean fluorescence
intensityd
M1
M1
M2
M4
M5b
M4
M2
89
93
45
90
100
21
87
1.87
2.24
2.28
1.45
3.21
1.71
1.09
Be
Sample
FIGOf
Histopathologyf
CALO
II B
INBL
IV B
pleomorphic nuclei, small
eosinophilic cytoplasm
metastatic, pleomorphic
nuclei, and hyperchromatic
a
mSF Mean fluorescence
intensityd
3.39
5.42
AML samples were collected at diagnosis as indicated in “Materials and Methods.”
M1 identifies AML without maturation, M2 with atypical maturation along the
granulocytic lineage, while M4 and M5 exhibit some monocytic features.
c
Percentage of blasts in the peripheral blood prior to cell separation.
d
The mean fluorescence intensity represents the ratio of fluorescence intensity of cells
that were stained with the monoclonal anti-mSF (7H6), as compared with a negative
control stained with an irrelevant isotype matched mAb (MAR18.5). A ratio ⬎1 indicates
that the cells are positive for mSF.
e
Epidermoid cervical carcinomas. Samples were collected at diagnosis as described in
“Materials and Methods.” Immunofluorescence for mSF was performed on established
cell lines.
f
FIGO staging system (Fédération Internationale des Gynaecologistes et Obstetristes).
The histopathology was assessed on H&E-stained tissue sections.
b
containing 5% FCS and 0.05% Na azide (Sigma Chemical Co., St. Louis, MO),
as well as 10% normal goat serum (Sigma Chemical Co.). Negative controls
were labeled with an irrelevant isotype-matched mAb. For the detection of the
SF receptor on cells, AML cells were labeled with a mouse antihuman c-Kit
(1:10; Caltag). The cells were washed and labeled with a second antibody
(PE-coupled sheep antimouse, 1:40; Sigma Chemical Co.). Dead cells were
excluded from the analysis on the basis of staining with propidium iodide as
well as forward and side scatter properties.
Establishment of Two Cervical Carcinoma Cell Lines. Among 60 different samples of cervical carcinoma, we established two cervical cancer cell
lines from surgical tumors in Mexican patients diagnosed with epidermoid
cervical carcinoma composed of nonkeratinized large cells from metastatic
(type IVB, named INBL) and nonmetastatic tumors (type IIB, named CALO).
Both patients were untreated at the time of tumor excision. The samples were
from the Mexican Institute of Social Security, (Clinic 8; Mexico City, Mexico)
and the National Cancer Institute (Mexico City, Mexico). Samples were
obtained and handled according to the National Cancer Institute guidelines. To
establish cervical carcinoma cell lines, the tumor was cut into small pieces and
enzymatically disaggregated with a mixture of collagenase (0.01%, Sigma
Chemical Co.) and trypsin (0.025%, Sigma Chemical Co.) for 45 min. Cells
were then washed in PBS and incubated in culture medium (RPMI; Life
Technologies, Inc.) supplemented with FCS. Both cell lines were maintained
in culture for ⬎50 passages. Cells were subcultured twice weekly at 0.5–
1 ⫻ 105 cells/ml in IMDM/10% FCS. Both cell lines are HPV18-positive (34,
35). As a prophylactic measure, parental cell lines were seeded into medium
with Ciprofloxacin HCl (Miles Inc., Kanakee, IL) at 50 ␮g/ml for 14 days to
avoid Mycoplasma.
Culture Conditions. AML blasts were plated in 96-microwell plates (Linbro; Flow Lab, McLean, VA) at 104 cells/well in 100 ␮l of IMDM (Life
Technologies, Inc.) supplemented with 10% FCS (Life Technologies, Inc.) and
1% methylcellulose (Fluka, Ronkonkoma, NY) as described previously (13).
Where indicated, serum-free cultures were performed in the presence of BSA
(20 mg/ml; Sigma Chemical Co.) and iron-saturated transferrin (30 ␮g/ml;
Hoechst, Pharmaceuticals Inc., Heidelberg, Germany) in OPTI-MEM medium.
CALO and INBL cell lines were seeded at 1.5 ⫻ 104 cells/ml, cultures were
maintained for 5 days. Cells were pulsed with tritiated thymidine (1 ␮Ci/well;
Amersham, Arlington Heights, IL) for 3 days before cell harvest. Cells were
then collected and retained on fiberglass filters (Schleider & Schuell, Keene,
NH), which were sequentially washed with 4 ml of PBS, 4 ml of 10%
trichloro-acetic acid, 4 ml of H2O, and 4 ml of methanol. Filters were dried
before liquid scintillation (EcoLume; ICN, Costa Mesa, CA) counting.
Karyotype. Exponential growing cells were incubated for 4 h in colchicine
(1 mg/ml, Sigma Chemical Co.). Cells were then trypsinized, centrifuged, and
allowed to swell in hypotonic solution for 12 min. The cells were then fixed in
2 ml of methanol:cold acetic acid (3:1) for 10 min, washed, resuspended in 0.5
ml of fixative solution, and dropped onto a cold slide. The slides were stained
with Giemsa, and chromosomes were visualized and counted. For each cell
type, 200 mitosis were scored.
Analysis of Phosphotyrosine-containing Proteins. One-million cells
were washed once with ice-cold PBS in a microfuge tube. Cell pellets were
rapidly resuspended in 40 ␮l of lysis buffer [20 mM Tris-HCl (pH 8.0), 132 mM
NaCl, 2 mM ethylene diamine tetra-acetic acid, 100 ␮M vanadate, 1 mM
phenylmethyl sulfonyl fluoride, 1% Triton X-100, and 10% glycerol] on ice.
Forty ␮l of 2 ⫻ Laemmli sample buffer [120 mM Tris (pH 6.8), 2 mM urea, 100
mM DTT, 10% vol/vol/glycerol, and 0.001% wt/vol bromphenol blue] was
immediately added with vortexing, and samples were boiled for 3 min. Fifty ␮l
of sample and molecular weight markers (Sigma Chemical Co.) were resolved
by electrophoresis on a vertical 7.5% SDS-polyacrylamide slab gel. After
electrophoresis, proteins were electroblotted onto nitrocellulose membranes.
Nonspecific binding sites were saturated by 1-h incubation at room temperature in TBS [10 mM Tris-HCl (pH 7.5) and 0.9% NaCl], containing 3% BSA
and 0.01% sodium azide, according to the recommendation of the manufacturer (DuPont, Boston, MA). Binding of 1 ␮g/ml of the primary murine
antiphosphotyrosine mAb (Boehringer clone 3-365-10) in TTBS and 2%
BSA-0.01% sodium azide, was performed overnight at room temperature.
Membranes were subsequently thoroughly washed with TBS and TTBS and
reacted with a rabbit antimouse (1:3000, Sigma Chemical Co.) followed by a
rat antirabbit coupled to alkaline phosphatase diluted 1:750 in TBS and 2%
BSA for 1 to 2 h at room temperature. After extensive washing in TTBS ⫻ 4
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and TBS ⫻ 2, membranes were transferred to a visualization solution composed of 10 ml alcaline phosphatase buffer [100 mM Tris-HCl (pH 9.5), 100
mM NaCl, and 5 nM MgCl2] containing 33 ␮l of nitroblue tetrazolium (50
mg/ml in 70% dimethyl formamide) and 66 ␮l of 5-bromo-4-chloro-3-indolyl
phosphate (50 mg/ml in 100% dimethyl formamide). Membranes were soaked
for 30 s to 1 min, and the reaction was stopped with water.
Immunoprecipitation. Exponentially growing CALO and TF-1 cells were
washed and incubated in OPTI-MEM (Life Technologies, Inc.) containing
1.0% BSA (Life Technologies, Inc.) for 24 h at 37°C to deprive cells from
growth factors; TF-1 cells were deprived from GM-CSF for 6 h. Before
stimulation, the cells were harvested by trypsinization (0.1%), washed, and
resuspended in appropriate medium. Tyrphostins 1 and B42 (10 ␮M) were
added, incubated for 5 min, stimulated with SF for 3 min, and rapidly pelleted
at 5,000 rpm for 5 min in a microfuge (Eppendorf). Cells were resuspended in
ice-cold lysis buffer [1% Triton X-100, 5 mM EDTA, 1 mM KCl, 140 mM
NaCl, 2 mM MgCl2, 50 mM Tris (pH 7.4), 1 mM phenylmethylsulfonyl fluoride,
1 mM NaF, 1% aprotinin, 1 ␮M leupeptin, and 100 ␮M Na3VO4] for 15 min.
Lysates were clarified by centrifugation at 13,000 rpm at 4°C for 15 min. Total
protein content of the lysate was determined by the Bio-Rad protein assay
(Bio-Rad, Hercules, CA).
Total lysate containing the same amount of proteins was incubated with
protein A-Sepharose (Life Technologies, Inc.) coupled previously to a specific
mouse antihuman c-Kit antibody (Boehringer-Mannheim, Mannheim, Germany; at 1 ␮g/ml) for 2 h at 4°C. Immunoprecipitated proteins were washed
5 times with lysis buffer, resolved by 7.5% SDS-PAGE, and transferred to
nitrocellulose membrane.
Western blots were analyzed using a mouse antiphosphotyrosine antibody
(clone 3–365-10; Boehringer-Mannheim) at 1 ␮g/ml, and proteins were visualized by alkaline phosphatase-conjugated antibodies system (Sigma Chemical
Co.). Substrates were nitroblue tetrazolium (8 mg/ml) and 5-bromo-4-chloro3-indolyl phosphate (1.6 mg/ml; Sigma Chemical Co.).
Antisense Oligonucleotide Uptake and Stability. Oligodeoxynucleotides
were 5⬘-labeled with 5⬘-{␥-[35S]thio} ATP (1276 Ci/mmol; Amersham) with
bacteriophage T4 kinase and purified by polyacrylamide gel electrophoresis.
AML blasts were incubated with 5 ⫻ 105 cpm of 35S-labeled oligonucleotide
at 4 ⫻ 106 cells in 0.5 ml of IMDM supplemented with 10% FCS at 37°C. At
the indicated times, cells were collected by centrifugation (3 mn, 15,000 ⫻ g),
washed twice in PBS, and the cell pellet was lysed in 0.1 ml of TBS [10 mM
Tris (pH 7.4) and 150 mM NaCl] with 1% NaDodSO4, and then extracted with
phenol. The aqueous phase was saved and the phenol re-extracted with water.
The combined aqueous phases were lyophilized, redissolved in 80% deionized
formamide, and analyzed in a denaturing 20% polyacrylamide gel.
For the AS-SF, a 20-mer oligonucleotide was designed that starts at the
translation initiation codon. A control oligonucleotide was synthesized in
which the sequence was shuffled:
AS-SF, CAA GTT TGT GTC TTC TTC AT
PAKL, TTG TTT ACT GCG TCT ACT AT
Whole Cell ELISA. Translation of Sl into mSF was quantitated by whole
cell immunosorbent assay. AML, CALO, and INBL cell lines were labeled for
30 min at 4°C with the mAb antihuman SF (7H6; 1 ␮g/106 cells). The cells
were washed 3 times with PBS/BSA 1% and labeled with a second antibody
(peroxidase-coupled goat antimouse, 1:1000; Sigma Chemical Co.) for another
30 min. The cells were washed 3 times, and cell-bound antibodies were
revealed with a 100-␮l o-phenylenediamine (1 mg/ml) in phosphate buffered
saline plus 14 ␮l of 30% H2O2. After 5 min (AML blasts) and 10 min (CALO
and INBL cells) at 37°C, the reaction was stopped by addition of 50 ␮l of 4 N
sulfuric acid. Then the cells were centrifuged, and the absorbance of the
cleared supernatants was read with an ELISA plate reader at 490 nm.
The TUNEL Reaction. AML (1 ⫻ 106 cells), CALO, and INBL (5 ⫻ 105
cells) were analyzed for apoptosis using a quantitative DNA fragmentation
assay, the TUNEL reaction. Briefly, cells were fixed with formaldehyde for 5
min at room temperature and permeabilized with 0.1% Triton X-100 and 0.1%
sodium citrate at 4°C for 2 min. Cells were then incubated for 1 h at 37°C with
0.3 nmol of biotin-16-dUTP (Boehringer-Mannheim), 3 nmol of dATP (Pharmacia, Uppsala, Sweden), 20 units of TdT (Pharmacia) in TdT buffer (0.1 M
potassium cacodylate pH 7.2, 2 mM CoCl2, 0.2 mM dithio threitol; Life
Technologies, Inc.) in a total reaction volume of 50 ␮l. The reaction was
stopped by the addition of 0.5 M EDTA. DNA from apoptotic cells that
contained free 3⬘-hydroxy ends were labeled by the TdT with biotinylated
dUTP, which was then revealed with streptavidin-FITC (Amersham, Buckinghamshire, England). After washing, the cells were cytosmeared and mounted
in Vectashield for counting.
RESULTS
Establishment of Human Cervical Carcinoma Cell Lines: Density-dependent Growth. Both CALO and INBL cells form cobblestone areas in FCS-containing medium. Karyotype analysis (Fig. 1A)
revealed that both cells were aneuploid, with CALO cells numbering
on average 50 chromosomes with a range of 32– 62 and INBL cells
numbering on average 50 chromosomes with a range of 48 – 66. Cell
cycle analysis by flow cytometry also confirmed the presence of an
aneuploid cycle, which was 17.1% for CALO and 23.7% for INBL
(data not shown). These observations are consistent with previous
work indicating pronounced chromosomal imbalances in primary
cervical carcinomas and cell lines (34).
We initially observed that cultures seeded at low density grew very
slowly with a long lag time, whereas cultures seeded at high cell
density propagated with a doubling time of 3– 4 days. Limiting
dilution analysis was therefore performed and revealed two distinct
growth patterns for both CALO and INBL cells (Fig. 1B). At low cell
concentrations, growth was extremely slow, whereas at concentrations
⬎15,000 cells/ml, the growth curve showed a much steeper slope.
This growth pattern was reproducibly observed, even after 50 passages. In contrast, HeLa cells, a cervical carcinoma cell line, exhibited
Fig. 1. Aneuploidy and density-dependent growth of two cervical cancer cell lines,
CALO and INBL. A, aneuploidy in CALO and INBL. For each cell line, 200 mitosis were
scored. B, density-dependent growth. CALO and INBL cells were plated at concentrations
varying from 2,000 to 64,000 cells/ml in 24-well plates for 5 days. Cells were pulsed with
[3H]thymidine (1 ␮Ci/well) for 3 days before harvesting. HeLa cells were plated at
concentrations varying from 500 to 4,000 cells/ml (n, number of representative experiments for each cell line). C, surface expression of HER-2 and c-Kit. CALO, INBL, and
HeLa cells were analyzed for surface expression of HER-2 (top panel) and c-Kit (bottom
panel) by flow cytometry. Cells labeled with an irrelevant isotype matched mAb
(MAR98) served as negative controls. Unlabeled cells were also analyzed for autofluorescence (data not shown). TF-1 cells served as a positive control for c-Kit and MCF7, a
breast carcinoma cell line for HER-2, respectively.
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a single accelerated growth pattern similar to the second proliferative
growth curve of CALO and INBL cells. Furthermore, conditioned
medium harvested from cultures seeded at high densities did not
accelerate the growth of cultures seeded at low densities (data not
shown). These results indicate that for the two cervical carcinoma cell
lines that we established, CALO and INBL, cell growth is densitydependent, consistent with a requirement in cell interaction for propagation in culture.
Because HeLa cells express HER-2 and primary cervical carcinomas express c-Kit, we investigated the surface phenotype of CALO
and INBL cells. As shown in Fig. 1C, all three of the cervical
carcinoma cell lines expressed HER-2 at comparable levels. In contrast, CALO and INBL expressed high levels of c-Kit (Fig. 1C, left
and middle panels) comparable with those found on the CD34⫹
hemopoietic cell line TF-1 (data not shown), whereas HeLa cells were
negative for c-Kit (Fig. 1C, right panel). We next investigated their
growth factor requirement. Cell growth was clearly dependent on
FCS, and serum withdrawal resulted in growth arrest, cell shrinkage,
and cell death through a process resembling apoptosis (Fig. 2A).
Interestingly, soluble SF added at 323 pM could substitute for FCS to
sustain growth in both INBL and CALO cells (Fig. 2A). In these cells,
soluble SF induced the tyrosine phosphorylation of c-Kit (Mr 145,000)
as identified by immunoprecipitation and of two other proteins of Mr
95,000 and 120,000 (Fig. 2, B and C). Treatment with tyrosine kinase
inhibitors (Tyrphostin B42) prevented c-Kit tyrosine phosphorylation
(Fig. 2C) and induced apoptosis in both cell types, as assessed by the
TUNEL reaction (Fig. 2D), whereas Tyrphostin1, an inactive analogue, did not affect cell viability. Both Tyrphostin B48 and Tyrphostin B42, shown previously to inhibit c-Kit signaling (33), induced
apoptosis to the same extent, suggesting that SF is a survival factor for
cervical carcinoma cells as documented for AML blasts.
c-Kit and mSF are Coexpressed in Cervical Cancer Cell Lines
and AML Blasts. Our results indicate that CALO and INBL cells
express functional c-Kit receptors in the same way as primary AML
blasts (11, 12). Therefore we addressed the question of whether these
cells coexpress c-Kit and mSF that could contribute to the process of
density-dependent growth (Fig. 1B; Ref. 12). A mAb specific for
human SF brightly stained 3T3 cells that were engineered to express
mSF (p220; Fig. 3B), whereas parental NIH-3T3 cells and TF-1 cells
were negative for mSF. Interestingly, CALO, INBL, and HeLa cells
expressed detectable levels of mSF (Fig. 3A). Six of seven primary
AML samples expressed mSF to various extents (16 –90%; Fig. 3B
and data not shown). Samples 32, 45, and 55 stained most brightly and
were classified as M1, M2, and M5, respectively. There was no
obvious correlation between mSF levels and FAB classification.
The surface marker CD34 is expressed in AML stem cells (36). As
the cells differentiate, they acquire myeloid surface markers such as
CD13 and loose CD34 expression. Because mSF is not detected in all
of the cells, we addressed the question of whether mSF expression is
a property of leukemic progenitors and/or of differentiating leukemic
cells. Double labeling indicated that both CD34⫹ and CD34⫺ AML
blasts expressed mSF (Fig. 4). Furthermore, most mSF⫹ cells are also
CD13⫹. Thus, mSF is expressed by leukemic progenitors and differentiating leukemic cells, consistent with the fact that expression of
mSF is not restricted to a FAB subtype.
Antisense Inhibition of mSF in AML Blasts and Cervical
Cancer Cell Lines. To address the significance of mSF expression in
these tumors, we sought to suppress mSF production through the use
of phosphorothioate-modified oligonucleotides. We first determined
the toxicity of control phosphorothioate oligonucleotide and found
that concentrations ⬍10 ␮M were well tolerated by INBL and CALO
cells, and ⬍4 ␮M by AML blasts (data not shown). Therefore, these
concentrations were chosen for subsequent experiments. Kinetic anal-
Fig. 2. Cervical cancer cell lines require FCS or SF for growth in culture. A, tissue
culture appearance of cervical cancer cell lines in the presence and absence of SF. CALO
and INBL cells were seeded at 15,000 cells/ml in IMDM/10% FCS. After overnight
adherence, the cells were deprived of growth factors in IMDM/BSA 1%, leading to lost
of cell viability 5 days later. In contrast, cells remain viable in IMDM supplemented with
FCS (10%) or SF (323 pM). B and C, SF induces c-Kit tyrosine phosphorylation in cervical
carcinoma cell lines. One-million cells were deprived of serum for 24 h in OPTI-MEM 1%
BSA and exposed to SF for 5 min. Total cell lysates were analyzed by Western blotting
for the pattern of protein tyrosine phosphorylation. Note that SF induces tyrosine phosphorylation of three major cellular proteins (arrows). Cell lysate was also immunoprecipitated with an anti-Kit, and tyrosine phosphorylation was revealed by immunoblotting
with an anti-phosphotyrosine mAb (3-365-10). D, addition of tyrosine kinase inhibitors
(tyrphostins) induces apoptosis. Cells were seeded at 32,000 cells/ml in culture medium
and SF (323 pM) with or without tyrphostins (10 ␮M), which were added after overnight
adherence. Five days later, apoptosis was determined by the TUNEL reaction. Tyr 1 is an
inactive analogue that was used as a negative control. Data are the mean of duplicate
cultures and are representative of n experiments; bars, ⫾ SD.
ysis indicated that the oligonucleotides were efficiently incorporated
into AML blasts and remained stable for ⱖ24 h (Fig. 5A), although at
8 h, the oligonucleotides were found in high molecular weight complexes. These complexes were resistant to detergent and heat denaturation, suggesting an irreversible association with cellular components. The level of inhibition of cell-associated SF was therefore
quantitated by whole cell ELISA. Cells were harvested every day
starting from 48 h after exposure. On day 2, half of the medium was
changed, and the cells were re-exposed to the same concentrations of
phosphorothioate-modified oligodeoxynucleotide. A 48-h treatment
with AS-SF was not sufficient to reduce mSF expression (data not
shown). After two cycles of 48 h of exposure to phosphorothioatemodified oligodeoxynucleotide, AS-SF induced a substantial decrease
in mSF levels in CALO and INBL cells, as determined by whole cell
immunoperoxidase (Fig. 5B) or by flow cytometry analysis (Fig. 5C).
The same antisense was also efficient in abrogating the production of
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Fig. 3. Expression of mSF in cancer cell lines. Cervical carcinoma
(CALO, INBL, and HeLa) cell lines (A) and primary AML blasts (B)
were analyzed for surface expression of mSF (7H6) by flow cytometry.
Cells labeled with an irrelevant isotype matched mAb (MAR18.5)
served as negative controls. p220, a 3T3 cell line that expresses human
membrane-SF, served as a positive control for mSF, whereas parental
NIH-3T3 cells and the hemopoietic cell line TF-1 serve as negative
controls. AML samples are described in Table 1. Data are representative of three or four independent experiments for CALO, INBL, and
AML 32.
mSF by the p220 3T3 transfectant (Fig. 5B) as well as in AML blasts
(Fig. 5D). In contrast, a PAKL did not affect mSF levels in these cells
(Fig. 5, B–D). To additionally determine the specificity of the AS-SF
oligonucleotide, we monitored the levels of another surface marker,
CD34, which is expressed on AML blasts. In contrast to SF, there was
no significant difference in CD34 levels in cells treated with AS-SF,
in the control oligonucleotide, or in parallel cultures that were left
untreated (Fig. 5D). Similarly, the AS-SF oligonucleotide did not
affect the level of HER-2 in CALO and INBL cells (data not shown),
indicating its specificity for mSF. For sample AML 53, there was
nonetheless some degree of apoptosis with the control oligonucleotide, consistent with [3H]thymidine incorporation studies (data not
shown), indicating a narrow window of efficiency above which the
oligonucleotide starts to have nonspecific toxicity (Fig. 5D). However, apoptosis was ⱖ2-fold higher with AS-SF. Taken together, our
observations clearly indicate that AS-SF specifically targets mSF in
AML samples as well as in gynecological tumors.
Antisense Inhibition of mSF in AML Blasts and Cervical
Cancer Cells Prevents Density-dependent Growth and Induces
Apoptosis. Our previous work indicates that autonomous cell proliferation in the culture of primary AML blasts occurs at high cell
concentrations, whereas at low concentrations, cell proliferation is
strictly dependent on an exogenous source of growth factor. Autonomous cell proliferation was first assessed by thymidine incorporation
Fig. 4. Both CD34⫹ and CD13⫹ cells express
SF. Primary AML blasts (AML 32) were stained
with CD34 or CD13 (IgG1) directly conjugated to
FITC and the antibody against SF (7H6, IgG2a)
followed by a goat antimouse IgG2a coupled to PE.
Control cells were stained with isotype-matched
irrelevant antibodies (MAR18.5, IgG2a and MR98,
IgG1). Data are representative of two independent
labelings with AML 32 blasts.
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Fig. 5. Stability and effect of AS-SF on mSF expression in AML blasts and CALO and INBL cells. A,
phosphorothioate-modified oligonucleotides are incorporated in AML blasts and remain stable for 8 h. AML
blasts were incubated with 32P-labeled antisense-SF oligonucleotide. Cells were harvested at the indicated
times, and cell extracts were resolved by SDS-PAGE. As
a control, the labeled oligonucleotide was loaded directly
onto the gel (C ). B, antisense inhibition of mSF expression in CALO, INBL, and p220 cells. Cells were seeded
at 15,000 cells/ml in culture medium and allowed to
adhere overnight before addition of oligonucleotides at
10 ␮M. mSF was quantitated by whole cell ELISA after
4 days of culture. 䡺, untreated cultures; u, cultures
treated with PAKL; f, cultures treated with AS-SF).
Data shown represent the mean ⫾ triplicate determinations and are typical of n experiments. For all three cell
types, the differences between control cell or PAKL- and
AS-SF-treated cells are highly significant (P ⬍⬍ 0.05),
whereas the differences between control cells and
PAKL-treated cells are not significant (P ⬎ 0.05). C,
AS-SF reduces mSF but not CD34 expression in AML
blasts. Cells (1 ⫻ 106) were seeded in the presence of 4
␮M of oligos and 40 pM of GM-CSF for 4 days. mSF and
CD34 were determined by whole cell ELISA assay. Data
shown represent the mean of triplicate determinations
and are typical of 2 experiments; bars, ⫾ SD. For both
samples AML 32 and AML 34, the differences between
control cells or PAKL and AS-SF-treated cells are highly
significant (P ⬍⬍ 0.05). In contrast, the differences between control cells and PAKL-treated cells are not significant (P ⬎ 0.05) for AML 34, and the value is at the
limit of significance (P ⫽ 0.04) for AML32.
and was attenuated by treatment with AS-SF in a dose-dependent
fashion (Fig. 6A). In contrast, the effect of the control oligonucleotide
was not significant in the dose range tested. Similarly, blast progenitors, assayed through their capacity to form colonies in vitro in
methylcellulose cultures either in the absence of growth factors or in
GM-CSF-containing cultures, were significantly decreased after treatment with the AS-SF oligonucleotide but not the control oligonucleotide. To additionally support the specificity of antisense inhibition of
cell-associated SF, we addressed the question whether addition of an
excess of soluble SF could compensate for decreased mSF expression
by the cells (Fig. 6A). We have shown previously that soluble SF can
enhance colony formation supported by GM-CSF. Under these conditions, growth suppression induced by the AS-SF oligonucleotide
was relieved, suggesting that the antisense was specific for mSF.
Because autonomous growth occurs under conditions favoring cellcell interaction, our results suggest that mSF contributes to cell
interaction in the culture of AML blasts.
A major function of SF is to suppress apoptosis (33). We therefore
addressed the question of whether decreasing mSF expression in
AML blasts would cause apoptosis as assessed by the TUNEL reaction. Data shown in Fig. 6, C and D, indicate that the AS-SF oligonucleotide induced apoptosis in both AML samples at levels that were
significantly higher than those observed with the control oligonucleotide, consistent with a critical role for mSF in sustaining cell survival.
We next addressed the role of mSF in the growth of cervical
carcinoma cells in vitro (Fig. 7) in FCS-containing cultures. Treatment with the control oligonucleotide did not significantly affect the
growth of CALO or INBL cells that remained adherent (Fig. 7A),
viable (i.e., TUNEL⫺; Fig. 7B), and were able to sustain DNA
synthesis as determined by thymidine incorporation (Fig. 7C). In
contrast, AS-SF oligonucleotides caused the cells to apoptose and
detach. Furthermore, antisense inhibition reduced thymidine incorporation to levels observed in cultures initiated at low cell density (Fig.
7C). Finally, HeLa cells that do not exhibit density-dependent growth,
as shown in Fig. 1B, were not affected by the oligonucleotide (data not
shown), consistent with the lack of c-Kit expression on these cells.
Together, our results indicate that mSF contribute to the densitydependent growth of cervical carcinoma cell lines and of AML blasts.
DISCUSSION
Cell interaction is a process common to cancers of diverse
origins. Cervical carcinomas and leukemic cells are shown here
and elsewhere (11, 12) to coexpress mSF and c-Kit. In vitro culture
assays for AML blasts revealed the importance of cell interaction
in the process of autonomous growth (12). Interestingly, only
irradiated cells or membranes isolated from AML blasts (37) could
substitute for cell interaction in suspension culture. Indeed, our
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Fig. 6. Antisense inhibition of mSF induces
apoptosis in AML blasts and prevents cell proliferation. A, AML blasts were plated in suspension
culture at 1 ⫻ 104 cells/well together with indicated
concentrations of oligonucleotides. Thymidine incorporation was determined on triplicate cultures as
described in “Materials and Methods” and are representative of n experiments. The differences between cultures exposed to SF-AS and PAKLs are
significant at 1 and 2 ␮M (P ⬍ 0.05). B, the effect
of oligonucleotides was evaluated on colony formation and cell survival. AML blasts were seeded
at a concentration of 104/well in IMDM/10% FCS
for colony formation in the presence or absence of
GM-CSF (40 pM) and oligonucleotides (4 ␮M). 䡺,
untreated cultures; u, cultures treated with PAKL;
f, cultures treated with the AS-SF. Data shown
represent the mean of 5 replicate cultures and are
typical of n independent experiments; bars, ⫾ SD.
The differences between AS-SF-treated and PAKL
or control cells maintained with GM-CSF or in the
absence of growth factors are highly significant
(P ⫽ 0.05), whereas all other differences are not
significant (P ⬎ 0.05). C and D, apoptosis was
determined by TUNEL reaction and percentage of
positive cells was calculated from a total number of
220 cells. The photographs shown in C were taken
at ⫻400. 䡺, AML 53 and u, AML 32. Data shown
are representative of n experiments.
attempts at replacing irradiated cells or membranes or at limiting
cell concentrations by soluble growth factors such as IL-1 (38),
IL-6 (39), tumor necrosis factor-␣ (40), and IFN-␥ (41) were not
successful. These results lead us to hypothesize that the presence of
membrane-bound growth factors could mediate cell-cell interaction in AML. Our previous work indicated that colony formation
as a function of cell concentration (12) was better described by a
model with two limiting parameters, the colony-forming cell itself
and an “interacting” cell. Interestingly, addition of high concentrations of soluble SF was sufficient to alleviate the requirement
for cell interaction. These observations led us to investigate the
possibility that these leukemic cells express mSF, which could
participate in the process of cell-cell interaction. In the present
study, we show that both undifferentiated (FAB M1, CD34⫹) and
differentiating (FAB M4/5 and CD13⫹) AML blasts express mSF,
shown here through an antisense strategy to be important in the
process of cell-cell interaction in AML.
There is evidence to suggest that c-Kit and SF are also involved in
the pathogenesis of nonhematological malignancies. Gynecological
tumors (22), colon tumor cell lines (23), small cell lung cancers (26,
42), and testicular tumors (43) have all been shown recently to
coexpress c-Kit and SF. To confirm the significance of these findings,
a mutation of the SF gene in a laboratory mouse, the Steel-Dickie
mutation was introduced into a line of HPV-E6E7 transgenic mice
that develop testicular tumors with full penetrance. In Steel-DickieE6E7 transgenic mice, tumorigenesis was initiated, but the tumor load
was markedly reduced compared with transgenic mice carrying wildtype Sl alleles, showing that c-Kit activation by SF is essential for
testicular tumorigenesis, especially in tumor promotion. These transgenic mice provide a useful in vivo model implicating growth factor
autostimulation in carcinogenesis (18, 19). Various W mutations (W,
Wv, and W42) were also introduced into the transgenic mouse strain
carrying HPV oncogenes in which c-Kit/Steel-mediated tumorigenesis occurs with high incidence. In all of the transgenic strains carrying
a W mutation, c-Kit deficiency affected the tumorigenic process to
various degrees. Tumor development was markedly suppressed in
transgenic strains carrying kinase defective mutations (Wv and W42) in
a heterozygous condition. In null-type (W) heterozygous transgenic
mice, tumorigenesis was suppressed to a lower level. Moreover,
minimal focal lesions or, in some cases, no focal lesion were found in
the testes of W/Wv compound heterozygous transgenic mice, showing
a close relationship between tumor cell growth and the degree of c-Kit
inactivation. In this mouse model of testicular tumors, it is not clear,
however, whether mSF-cKit interaction is important in paracrine or
autocrine stimulation. Furthermore, there was no direct evidence for a
role of c-Kit and mSF in cervical cancers, the most prevalent cancer
in women in Latin America.
The role of mSF-Kit in AML blasts and in a mouse model of
testicular tumors led us to address the role of this ligand-receptor pair
in tumors of the cervix. Previous work indicates that transcripts for SF
but not c-Kit are found in the commonly used cervical carcinoma cell
lines (HeLa, Caski, and SiHa). We show here that HeLa cells do not
require cell interaction for propagation in culture, possibly because of
the fact that the cells have been extensively expanded in culture and
may have acquired additional mutations bypassing this property. In
contrast, the two cervical carcinoma cell lines that we have established, CALO and INBL, have retained the characteristics of most
primary tumor cells, i.e., growth factor dependence for cell survival
and density-dependent cell proliferation. Furthermore, we show that
these two cell types coexpress mSF and cKit and that mSF is an
important determinant of cell survival and cell-cell interaction in
cervical carcinomas.
Tumorigenesis is a multi-hit process that involves several independent events, and evidence shown here and elsewhere suggests that
autocrine growth factor production (1) is one of the more common
perturbations, responsible for the final stages of uncontrolled growth
and survival. Initially, autocrine stimulation was defined as the capacity of tumor cells to secrete and respond to growth factors (1). This
concept later evolved to include a “private” autocrine loop in which
growth factors interact with their receptors intracellularly (2). Our
observations suggest that autocrine stimulation may frequently be a
juxtacrine process mediated by membrane-linked growth factors.
Such a process may in fact be amplified by signaling through additional “ligand-receptor”-type interaction belonging to the superfamily
of adhesion molecules. Consistent with this hypothesis, previous work
indicates that mSF is more potent than soluble SF in maintaining the
survival of multipotent hemopoietic stem cells in vitro (7). Therefore,
we propose that juxtacrine stimulation by membrane-linked growth
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Fig. 7. Antisense inhibition of mSF induces apoptosis in CALO and INBL cells and prevents densitydependent growth. A, CALO and INBL cells were
plated at 15,000 cells/ml with AS-SF or PAKL as
described in Fig. 5B. After 5 days of culture, a representative field was photographed from control and
oligonucleotide-containing cultures. B, apoptosis was
examined by TUNEL reaction. C, [3H]thymidine incorporation. Oligonucleotides (10 ␮M) were added after overnight adherence, and thymidine incorporation
was determined as described in Fig. 1B. 䡺, untreated
cultures; u, cultures treated with PAKL; f, cultures
treated with the AS-SF. Data shown are typical of two
independent experiments and in C, represent the mean
of triplicate determinations; bars, ⫾ SD. Differences
between AS-SF and PAKL control-treated cells are
highly significant (P ⫽ 0.05).
factors may underlie the process of cell-cell interaction and densitydependent growth in cancers, which may be more common than
anticipated previously.
As a consequence of autocrine stimulation in cancers, receptor
antagonists became potential targets in cancer therapy. For example,
IL-1R antagonist and soluble IL-1R were used in chronic myeloid
leukemia aimed at inhibiting the action of autocrine IL-1␤ (44).
However, antagonists are described for only a few growth factor
receptors, and IL-1R does not appear to mediate cell-cell interaction.
The antisense strategy described here aimed at interrupting mSF
production by leukemic cells, and cervical carcinomas may prove
useful as a novel adjuvant therapy with improved specificities. HPV16
E6/E7 antisense oligonucleotides were shown to decrease the proliferation of HPV-positive cells, albeit with low efficacy (45). It may
therefore be possible to achieve high efficacy and specificity by
targeting two genetic events in HPV-positive gynecological tumors,
the HPV E6/E7 oncogenes and mSF. The in vivo efficacy of a targeted
genetic therapy was shown recently in a clinical trial with patients
suffering from advanced cancers. These patients were treated with a
phosphorothioate antisense oligodeoxynucleotide directed to the 3⬘
untranslated region of the c-raf-1 mRNA, resulting in significant
reductions of c-raf-1 expression by day 3 of treatment. Furthermore,
the time course and depletion of c-raf-1 message in peripheral blood
mononuclear cells paralleled the clinical benefit in two patients (46).
Given that normal hemopoietic stem cells require SF in vivo, a
strategy based on AS-SF will require accurate determination of its
therapeutic window. The possibility of xenografting human AML
blasts (36) and human cervical carcinoma cells (47) in immunedeficient mice and the availability of mouse models of testicular
tumors (48) will allow for a preclinical evaluation of AS-SF on tumor
burden in vivo and for its effect on normal hemopoiesis.
In summary, our results indicate that in both AML blasts and
cervical carcinomas, exponential growth is dependent on cell concentrations suggesting a requirement in cell interaction. Antisense inhibition of cell-associated SF blocks this interaction by disrupting cell to
cell contact mediated by the association of c-Kit with membranebound SF.
ACKNOWLEDGMENTS
We thank Dr. Isabel Soto-Cruz for her help in establishing the immunoprecipitation and Western blot technique, Ranulfo Pedraza and Jose Echavaria for
technical assistance, Christian Charbonneau and Nathalie Tessier for the imaging and flow cytometry service, respectively, and Viviane Jodoin for secretarial help.
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Implication of Tyrosine Kinase Receptor and Steel Factor in
Cell Density-dependent Growth in Cervical Cancers and
Leukemias
Julio R. Caceres-Cortes, Jose A. Alvarado-Moreno, Kazuo Waga, et al.
Cancer Res 2001;61:6281-6289.
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