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Regular Articles |
From the Department of Dermatology and Emory Skin Disease Research
Core Center*
and the Department of
Pathology,
Emory University School of
Medicine, Atlanta, Georgia; the Laboratory for Surgical
Research,
Department of Surgery,
Harvard Medical School and Children’s Hospital, Boston, Massachusetts;
and the Department of Adult Oncology,§
Dana
Farber Cancer Institute, Boston, Massachusetts
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have established a two-step model for the development of malignant endothelial tumors by the sequential introduction of a temperature-sensitive SV40 large T antigen and activated H-ras into murine endothelial cells. The addition of activated H-ras causes a switch from cells that produce small quantities of angiogenic mediators to aggressive angiosarcomas, which produce high levels of angiogenic mediators. We have previously shown that treatment with wortmannin, a potent inhibitor of the PI-3-kinase pathway, resulted in down-regulation of the angiogenic mediators VEGF and matrix metalloproteinases, and decreased in vivo tumor size. However, inhibition of PI-3-kinase had no effect on the angiogenic antagonist tissue inhibitors of matrix metalloproteinases (TIMPs).11 In this report, we describe the effect of inhibition of the MAP kinase signal transduction pathway by both introduction of a dominant negative MAPKK gene into angiosarcoma cells that express SV40 large T antigen and H-ras, and treatment of angiosarcoma cells with a chemical inhibitor of MAPKK, PD98059. These studies show that inhibition of the MAP kinase pathway leads to decreased proliferation and morphological reversion to the untransformed phenotype, as well as greatly decreased growth in soft agar, yet cells remain highly tumorigenic in vivo. These results point to a role of the MAP kinase pathway in mediating soft agar growth and a tissue-specific effect of the MAP kinase pathway on tumorigenesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MS1 and SVR cells are derived from primary murine endothelial cells and were maintained in Dulbecco’s modified Eagle’s medium supplemented with 5% fetal calf serum.11 SVR cells were infected with retroviruses encoding ß-galactosidase (F. Boyce, Massachusetts General Hospital, Boston, MA) or a dominant negative MAPKK mutant A221 (C. Marshall, Institute of Cancer Research, London, UK). Both vectors encode puromycin resistance, and cells were selected in 2 µg/ml puromycin. ß-Galactosidase expression was confirmed by histochemical staining with x-gal,12 and expression of the dominant negative MAPKK gene was confirmed by growing individual clones and performing Western blot analysis with an antibody specific to rabbit MAPKK and phosphorylated MAPK. Protein extracts were prepared as described in Arbiser et al.11 Two clones with reduced expression of phosphorylated MAP kinase were selected for further study and were named SVRA221a and SVRA221b. Expression of the dominant negative MAPKK was confirmed using antibody 177 to rabbit MAPKK (C. Marshall). This antibody cross-reacts with endogenous murine MAPKK, so an authentic clone that expresses the gene would be expected to have elevated levels of total MAPKK (rabbit transgene and endogenous murine MAPKK), but diminished phosphorylated MAP kinase. A clone expressing ß-galactosidase was named SVRbag4 and was found to have identical expression of phosphorylated MAP kinase as parental SVR cells, and high level expression compared with MS1 cells, which do not contain activated H-ras. Both SVRA221a cells, which express the lowest quantity of phosphorylated MAP kinase, and SVRbag4 cells have been submitted to the American Type Culture Collection (Manassas, VA) for distribution.
In Vitro Proliferation Assays
We plated 10,000 cells of each cell type in 24-well dishes. The next day, the medium was replaced with fresh medium containing the inhibitors or vehicle controls. Cells were incubated at 37°C for 72 hours, and cell number was determined in triplicate using a Coulter Counter (Hialeah, FL). PD9805913 and LY29400214 were obtained from Calbiochem (San Diego, CA) and were reconstituted in dimethylsulfoxide (DMSO) to a final concentration of 5 mg/ml stocks.
Western Blotting
Cells were lysed in lysis buffer containing 20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1% (v/v) Triton X-100, 10% glycerol, 1 mmol/L EDTA, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mmol/L benzamidine, 1 mmol/L phenylmethylsulfonyl fluoride, and 1 mmol/L Na3VO4.
Protein concentration was determined by the Bradford assay using bovine serum albumin as a standard. Samples were treated with Laemmli sample buffer and heated to 90°C for 5 minutes before sodium dodecyl sulfate-polyacrylamide gel electrophoresis (National Diagnostics, Atlanta, GA) and transfer to nitrocellulose membranes. The membranes were then blocked with 5% nonfat dry milk in TBST and subsequently incubated with the appropriate antibody for immunoblotting. Anti-Erk2 monoclonal antibody was from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-phospho-MAP kinase polyclonal antibody was from Promega (Madison, WI).
Assays of Matrix Metalloproteinase Bioactivity
Cells were grown to approximately 75% confluence in Dulbecco’s modified Eagle’s medium supplemented with 5% fetal calf serum. After washing with phosphate buffered saline, medium was replaced with Cellgro Serumless medium (Mediatech, Herndon, VA). Wild-type cells were incubated in serum containing medium supplemented with 5 µg/ml PD98059 or an equal volume of DMSO before shifting into Cellgro medium containing either PD98059 or DMSO vehicle. In the case of SVRA221a or SVRbag4 cell lines, medium was shifted to Cellgro serumless medium for 24 hours before harvest.
Substrate gel electrophoresis (zymography) was conducted as described by us previously.11 Briefly, Type 1 gelatin was added at a concentration of 1 mg/ml to the standard Laemmli acrylamide polymerization mixture. Conditioned medium from equivalent numbers of cells was diluted 3:1 with sample buffer (10% sodium dodecyl sulfate, 4% sucrose, 0.25 mol/L Tris (pH 6.8), 0.1% bromphenol blue) and electrophoresed as previously described. At the end of electrophoresis, gels were rinsed in 2.5% Triton X-100 for 30 minutes, then incubated overnight in substrate buffer (50 mmol/L Tris, pH 8, 5 mmol/L CaCl2, 0.02% NaN3). The gels were stained with 0.5% Coomassie blue R-250 in acetic acid:isopropyl alcohol:H2O (1:3:6) and destained in the same buffer in which the Coomassie blue was prepared. Densitometry of destained areas was quantified using a Datascopy GS Plus scanner connected to Macintosh II computer with Macimage software (Xerox Imaging Systems).
Assay of TIMP Bioactivity
TIMP bioactivity was quantified using conditioned media from equal numbers of cells treated with either PD98059 or vehicle control, or equal numbers of stable cells expressing the dominant negative MAPKK (SVRA221a) or vector control (SVRbag4). A solid collagen film assay was performed using C14-labeled collagen as previously described by us.11
Assay of Anchorage-Independent Growth
Anchorage-independent growth was determined by adding equal numbers of viable cells to a plate containing 0.35% Noble agar in 10% fetal bovine serum containing media at a density of either 5 x 104 or 5 x 105 cells per 6-cm2 plate. The number of colonies formed in agar was recorded 2 weeks after plating the cells.15 The effect of the MAP kinase inhibitor PD98059 on soft agar formation of SVRbag4 cells was assessed by continuous treatment of SVRbag4 cells with either 50 µmol/L PD98059 or DMSO vehicle control for 4 days before trypsinization. Once trypsinized, the cells were placed into growth medium containing either 50 µmol/L PD98059 or DMSO vehicle for the entire soft agar assay.
In Vivo Tumorigenesis
SVRbag4 and SVRA221a,b (1 x 106) cell lines were injected into the flank of 6-week-old nude male mice obtained from Massachusetts General Hospital. Three weeks after tumors appeared, they were excised and fixed in both formalin and Carnoy’s fixative for hematoxylin and eosin staining. Tumor volume was measured using the formula (width x width x length) x 0.52, where width represents the shortest dimension.11
To determine whether the inability to grow is soft agar was lost after in vivo growth, tumors of SVRbag4 and SVRA221a,b were explanted into tissue culture; once confluent, cells were assayed for their ability to grow in soft agar as described above. Cells were analyzed for growth in soft agar and expression of MAPKK by Western blot once explanted from mice.
Cell Cycle Analysis
Nuclei from cultured cells were isolated and stained with propidium iodide using the CycleTest Plus DNA Reagent Kit (Becton Dickinson, San Jose, CA). Data acquisition and analysis were performed on a FACSort flow cytometer (Becton Dickinson) using ModFitLT version 2.0 software (Becton Dickinson).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SVR cells were infected with recombinant retroviruses encoding
either (i) a dominant negative MAPKK mutated at allele Ser-221
in the catalytic domain or (ii) ß-galactosidase, and
selected in puromycin. Resistant clones were screened by Western blot
analysis for expression of phosphorylated MAP kinase and for expression
of the rabbit MAPKK allele using antibody 177, which is specific
for rabbit MAPKK.16
Two clones expressing decreased levels
of phosphorylated MAP kinase were selected for further study. A
ß-galactosidase-expressing clone was selected as a control and showed
high levels of phosphorylated MAP kinase expression (Figure 1)
. This clone had identical expression
of phosphorylated MAP kinase compared with parental SVR cells (data not
shown).
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SVR cells have a spindle-type morphology, which is retained on
introduction of ß-galactosidase (Figure 2B)
, but treatment of parental SVR cells
with PD98059 led to increased cellular size and width (Figure 2C)
.
Similar morphological changes were seen with introduction of the MAPKK
mutant Ser-221 (Figure 2D)
. These morphological changes are
similar to endothelial cells (MS1 cells), which lack activated ras
activity (Figure 2A)
. Treatment of cells with the
phosphoinositol-3-kinase inhibitor LY294002 had no noticeable
morphological effect on SVR or SVRbag4 cells.
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Both cell lines were grown in the presence of a MAPKK inhibitor,
PD98059, and a phosphatidylinositol-3-kinase inhibitor LY294002 in
doses ranging from 0 to 10 µg/ml, and cell counts were performed 72
hours later. Introduction of the 221 allele of MAPKK led to a decrease
of nearly 75% in growth rate. Both cell types were susceptible to
growth inhibition by LY294002, the phosphatidylinositol-3-kinase
inhibitor, in a dose-dependent fashion, but the SVRA2221a cells were
relatively resistant to growth inhibition by PD98059. In contrast,
growth of control SVRbag4 cells was highly inhibited by PD98059 (Figure 3)
. No cytotoxicity was observed as a
result of treatment with either PD98059 or LY294002.
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Effect of MAPKK Inhibition on MMP and TIMP Bioactivity
Treatment of SVR cells with the low molecular weight inhibitor
PD98059 caused a modest increase in gelatinase activity in conditioned
media (Figure 4)
. A similar pattern of
enhancement of MMP bioactivity was observed in SVRA221a, which
expresses the dominant negative MAPKK gene, compared with the vector
control cells SVRbag4 (Figure 4)
.
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, acute
down-regulation of MAPKK by PD98059 resulted in up-regulation of TIMP
bioactivity, whereas stable down-regulation of MAPKK resulted in no
change in TIMP bioactivity (Figure 5)
.
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The overexpression of a dominant negative mutant form of MAPKK
suppressed ras-induced anchorage-independent growth in angiosarcoma
cells. MS1 endothelial cells transformed by H-ras and containing a
vector control (SVRbag4) display anchorage-independent growth as
measured by their ability to form colonies in soft agar (Figure 6
and Table 1
). Ectopic expression of a dominant
negative mutant form of MAPKK in the ras-transformed angiosarcoma cell
lines (SVRA221a,b) dramatically decreased the number of colonies
observed in soft agar, as compared with SVRbag4 (Figure 6
and Table 1
).
Similarly, treatment of wild-type SVR cells with PD98059 suppressed the
ability of these cells to grow in soft agar. The ability of the cell
lines to grow in soft agar correlated with the expression levels of
phosphorylated MAP kinase. These data suggest that activation of MAPKK
is required for ras-induced anchorage-independent growth of
angiosarcoma cells in soft agar.
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One million cells of each type were injected subcutaneously into
the right flank of nude male mice. The tumors derived from the cells
expressing the dominant negative MAPKK gave rise to rapidly growing
tumors that did not vary significantly in size from wild-type SVRbag4
tumors (Figure 7)
. The decreased
phosphorylation of MAPKK was also observed after in vivo
growth, thus ruling out the possibility that tumor growth in animals
was secondary to loss of the transfected gene (data not shown). The
finding of equal tumorigenesis of the clones of SVR cells is
surprising, in light of the greatly impaired ability of the SVRA221a,b
cell lines to form colonies in soft agar. The decreased ability of
cells containing the dominant negative MAPKK was preserved after
in vivo growth, as cells explanted from rapidly growing
SVRA221a,b tumors showed diminished growth in soft agar compared with
wild-type SVRbag4 cells. The average volume of the SVRbag4 tumors was
1225 mm3, of SVRA221a tumors, 2783
mm3, and of SVRA221b tumors, 1468
mm3. The tumor volume of SVRA221a and
SVRA221b did not differ significantly from that of SVRbag4.
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To determine whether the decreased growth rate of SVRA221a cells was due to decreased plating efficiency or was secondary to changes in cell cycle progression, cells were stained with propidium iodide and analyzed by flow cytometry. Wild-type SVRbag4 cells showed the following percentages of cells in G0-G1: 53.6%, G2-M: 4.3%, and S: 42.1%, whereas SVRA221a cells showed G0-G1: 59.4%, G2-M: 15.7%, and S: 24.9%. Thus, SVRA221a cells showed a decreased percentage of cells in S phase, suggesting that the decreased growth in vitro is due in part to slowing of cell cycle progression.
Maintenance of Phenotype after Tumorigenesis in Mice
To determine whether the dissociation of soft agar growth and
in vivo tumorigenesis was maintained, we explanted tumors
from animals bearing SVRbag4 and SVRA221a,b into tissue culture. Cells
were cultured from tumors, and the ability of these cells to form
colonies in soft agar was analyzed. Consistent with the phenotype
before implantation, SVRA221a,b were significantly impaired in their
ability to form colonies in soft agar, even after in vivo
growth (Table 2)
. In addition, high level
expression of transgene was observed in explanted cells, with A221a
exhibiting a sixfold increase and A221b showing a twofold increase in
total (transgene and endogenous) MAPKK compared with SVRbag4
(endogenous murine MAPKK; Figure 8
). This
is consistent with maintenance of the phenotype after in
vivo growth.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Primary animal cells and untransformed cell lines require a solid surface on which to attach, spread, and multiply, a phenomenon known as anchorage-dependent growth. These cells, if detached from solid supports, undergo apoptosis through a process termed anoikis.25 Researchers have taken advantage of the property of transformed cells to grow in suspension, using agar, to show the ability of viruses to transform cells in an anchorage-independent assay.26-29
A correlation exists between anchorage-independent growth and tumorigenicity in mice.15,26-29 Shin et al have demonstrated that other cellular phenotypes of transformation, such as cell density and growth in reduced serum are not strongly correlated with neoplastic growth in vivo.29 The ability of cells to grow in the absence of adhesion presumably reflects the ability of transformed cells to survive and grow in inappropriate locations in vivo.
In this report, using the various endothelial cell lines, we tested their transforming ability in vitro by a colony formation assay in soft agar. These results suggest that the endothelial cell line MS1, which is normally anchorage-dependent, requires the activation of MAPKK downstream of ras to be transformed in vitro. In contrast, SVRA221a,b cells, which demonstrate low levels of MAP kinase activation and poor growth in soft agar, are fully tumorigenic in vivo. A potential predictor of the effect of signal transduction inhibition in a tumor cell line may be the bioactivity of MMPs, enzymes required for tumor growth and metastasis.30 Inhibition of MAPKK in our system, by both the small molecular weight inhibitor PD98059 and a dominant negative MAPKK led to an increase in MMP bioactivity. Another MAPKK-related gene, MAPKK-4, has been implicated as a tumor suppressor gene in gastrointestinal malignancy,31 so pharmacological inhibition of MAPKK may be beneficial in some malignancies, but not in others.
Our findings, that introduction of a dominant negative MAPKK slows growth in vitro and inhibits soft agar growth, but not tumorigenesis, are surprising. However, one must take into account the different environments of these assays. Growth in vitro is partially a function of the cell to proliferate in two dimensions attached to a plastic surface. Growth in soft agar requires three-dimensional growth without attachment to physiologically relevant basement membrane proteins and host integrins. Growth in vivo involves three-dimensional growth in the presence of host basement membrane proteins and integrins. Our results suggest that down-regulation of MAP kinase may play a role in two- and three-dimensional growth in a solid area in the absence of host proteins and integrins, but is not required for in vivo growth. Reliance on the soft agar assay alone may lead researchers to overlook potentially important oncogenic events.
Our results have several implications. First, MAPKK is not likely to be the major signal transduction pathway in regulating tumorigenesis and angiogenesis in ras-transformed angiosarcoma. Recent data showing that overexpression of the catalytic subunit of PI-3-kinase results in avian angiosarcomas suggest that PI-3-kinase mediates the angiogenic switch in angiosarcoma.21 Second, growth rate in vitro cannot always be correlated with in vivo tumorigenicity. Control SVRbag4 cells grow much more rapidly than MAPKK-deficient SVRA221a cells in vitro, but are about equally tumorigenic. Consistent with our studies in mice, we have demonstrated that phosphorylated MAP kinase is elevated in benign endothelial neoplasms (hemangiomas and pyogenic granulomas), but shows very low level expression in malignant angiosarcoma.32 These findings suggest that high level expression of phosphorylated MAP kinase is not required for human angiosarcoma growth in vivo, consistent with the findings of this study. Examination of expression of phosphorylated MAP kinase may prove diagnostically useful in endothelial neoplasms. Finally, prior studies have concluded that anchorage-independent growth is synonymous with tumorigenesis, and, conversely, that loss of soft agar growth means cells are incapable of forming tumors. Our results suggest that this correlation is not perfect, and that in vivo studies are required to fully assess tumorigenesis. Knowledge of the effect of MAPKK inhibition on in vivo growth will be necessary to determine the type of tumor that will be most responsive to MAPKK inhibition. Finally, MAPKK inhibitors may be pharmacologically useful for some malignancies but may fail to suppress the growth of other malignancies, depending on tissue type.
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Acknowledgements |
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Footnotes |
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Supported by the Society for Pediatric Dermatology, the Dermatology Foundation, and the Thomas B. Fitzpatrick KAO Award (to J. L. A.), American Skin Association and National Institute of AMS grants R03AR44947 (to J. L. A.), and Emory Skin Disease Research Core Center P30 AR 42687 and KO8 AR02030 (to J. L. A.). M. A. M. is supported by American Cancer Society grant 83821. K. R. L. is supported by the Garrett B. Smith Foundation and the Leukemia and Lymphoma Society.
Accepted for publication September 8, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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