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Technical Advances |
From the Departments of Dermatology
* and Pathology,
Emory University School of Medicine and the Winship Cancer Institute, Atlanta, Georgia; the Department of Internal Medicine,
University of Washington School of Medicine, Seattle, Washington; the Departments of Adult Oncology
and Pediatric
Oncology,
Dana Farber Cancer Institute and the Harvard Medical School, Boston, Massachusetts; the Department of Dermatology,
¶ Stanford University School of Medicine, Stanford, California; the Department of Neurology,
|| Washington University School of Medicine, St.
Louis, Missouri; and the Department of Pediatrics/Medical
Genetics,
** University of Texas–Houston Division of Pediatric Hematology/Oncology, Houston, Texas
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, PPAR), and melanocytes
(microophthalmia, MITF), thus demonstrating that a
single cell type can exhibit all of these phenotypes. These cells
should serve as a valuable tool to elucidate signal transduction
pathways underlying renal angiomyolipomas.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tuberous sclerosis complex is the result of mutations in one of two genes, Tsc1 (hamartin),7 and Tsc2 (tuberin).7,8 The functions of these genes are not completely understood, however, they have been shown to functionally interact with each other.9-11 Two mammalian models for tuberous sclerosis complex have been studied. Eker rats have a retroviral insertion in the Tsc2 gene and develop renal cell carcinomas and splenic angiosarcomas.12 Mice heterozygous for a targeted mutation in the Tsc2 gene also develop renal cell carcinomas, but also have a high incidence of hepatic hemangiomas.13,14 However, neither animal model develops angiomyolipomas. To obtain further insight into signal transduction pathways in angiomyolipoma, we have generated a stable angiomyolipoma cell line by sequentially introducing SV40 large T antigen and human telomerase into tumor cells from a sporadic human angiomyolipoma. The techniques described here are generally applicable to any benign neoplasm. These studies may lead to increased knowledge of signal transduction abnormalities in angiomyolipoma and eventually to medical therapy for angiomyolipoma.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Viable angiomyolipoma tissue was obtained from a spontaneous renal angiomyolipoma during a total nephrectomy. The patient was a 63-year-old female with no history of tuberous sclerosis complex or physical stigmata characteristic of tuberous sclerosis according to the Gomez criterion. Tumor tissue was dissociated with sterile filtered collagenase type II (Worthington, Lakewood, NJ) in phosphate-buffered saline after manual dissociation through repetitive pipetting with a plastic pipette. Collagenase was neutralized with serum-containing media, and the cells were cultured in type II complete media (50/50 mixture of Dulbecco’s modified Eagle medium/Ham F12), supplemented with sodium selenite 5 x 10-8 mol/L, insulin 25 µg/ml, hydrocortisone 2 x 10-7 mol/L, transferrin 10 µg/ml, T3 (triiodothyronine) 1 x 10-9 mol/L, vasopressin 10 µU/ml, cholesterol 1 x 10-8 mol/L, ferrous sulfate 1.6 x 10-6 mol/L, epidermal growth factor 10 ng/ml, and 15% fetal bovine serum (supplied by Elizabeth Henske, Fox Chase Cancer Center, Philadelphia, PA). Two weeks after culture, cells were transfected with 40 µg of the plasmid pMKSVori-,15 which encodes the entire genome of SV40 except for a small deletion at the origin of replication, in the presence of 40 µl of lipofectamine (Life Technologies, Inc., Gaithersburg, MD) in 2 ml of complete serumless media (Cellgro, Herndon, VA). Clones were picked from the transfected cells, and one clone was selected for introduction of telomerase based on ultrastructural similarities with authentic angiomyolipomas.
Electron Microscopy
To determine the morphological characteristics of SV40-transfected clones, electron microscopy was performed. Cell pellets were immersed in 4% cacodylate-buffered glutaraldehyde. After fixation, the cells were washed in buffer, fixed in 1% OsO4 solution, dehydrated in graded ethanols and propylene oxide, and embedded in Embed-812 epoxy resin (Electron Microscopy Sciences, Fort Washington, PA). Thick sections (0.5 µmol/L) cut with glass knives were stained with Toluidine Blue. Ultra-thin sections (0.1 µmol/L) sections were cut with a diamond knife and mounted on 200-mesh copper grids, stained with uranyl acetate and lead citrate, and photographed with a Phillips EM201 electron microscope (Phillips, Marburg, Germany). One clone (SV7) that was ultrastructurally most similar to angiomyolipoma was selected for further characterization.
Activation of Telomerase in SV40-Transfected Cells
Because all of the SV 40-transfected cell lines entered senescence after prolonged passage, we decided to overexpress human telomerase into SV7 cells, because these cells ultrastructurally resembled angiomyolipoma tissue. An MMLV-based retroviral vector, LZRS16 containing the full-length cDNA for human telomerase reverse transcriptase (hTERT; Geron Corp., Menlo Park, CA) was used to activate telomerase as previously described.17 Briefly, hTERT-LZRS was produced in the Phoenix-packaging cell line after stable transfection and replication-deficient virus was stored at -80°C until ready for use. One day before infection, SV7 cells were plated at 60% confluency in 6-well dishes and on the day of infection, the cells were pre-incubated in Dulbecco’s modified Eagle medium containing10% fetal bovine serum with 5 µg/ml polybrene at 37°C for 10 to 15 minutes. Media was removed and viral stock containing 5 µg/ml of polybrene was added, followed by centrifugation (300 x g) for 1 hour at 32°C. Cells were then incubated for 5 to 6 additional hours at 32°C, followed by continuous passage in original growth media. Activation of telomerase in transduced cells was confirmed by a polymerase chain reaction (PCR)-enzyme-linked immunosorbent assay-based telomere repeat amplification protocol (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacture’s recommendations. hTERT(+) SV7 cells maintained telomerase activity for at least 40 population doublings and show no activity decrease at the time of this publication. These cells are now known as SV7 tert. SV7 tert cells have been submitted to the American Type Culture Collection for general distribution (ATCC CRL-2461).
Immunohistochemical Analysis of SV7 Tert Cytospins
We performed cytospins of SV7 tert cells to determine whether they demonstrated phenotypic characteristics of angiomyolipoma cells, namely expression of melanocytic and smooth muscle markers. SV7 tert cells were released from tissue culture flasks with trypsin, and trypsin was neutralized by centrifugation of cells through 9 ml of Dulbecco’s modified Eagle medium supplemented with 10% fetal calf serum. The resulting cell pellet was fixed in 10% buffered formalin and was embedded in paraffin. Cells were stained with antibodies for muscle-specific actin HHF-35, 1:100, (DAKO Corp., Carpinteria, CA), and microphthalmia transcription factor (MITF) C5+D5, 1:40, (Neomarkers). Negative controls consisted of substitutions of serum for primary antibody.
Molecular Analysis of MITF Expression in SV7 Tert Cells
Reverse transcriptase-PCR was performed using 1 µg of total RNA isolated from SV7 tert and control melanoma cells (501 mel) with Tth DNA polymerase (Roche Molecular Biochemicals) as suggested by the manufacturer using an MJ Research PCR apparatus (PTC-200). 5' primers were specific for A or M MITF isoform RNA. The 3' primer sequence was common to both RNA species. The M form primer comprises nucleotides 21 to 44 of the M form MITF message with the sequence 5'-CCT TCT CTT TGC CAG TCC ATC TTC-3'. The A form primer comprises nucleotides 170 to 193 of the A form message with the sequence 5'-TGA AGA GCC CAA AAC CTA TTA CGA-3'. The 3' primer comprises the complement of nucleotides 906 to 883 of the A form message with the sequence 5'-GAT CAA TCA AGT TTC CCG AGA CAG-3'. PCR products were visualized after 1% agarose electrophoresis by ethidium bromide staining. For immunoprecipitation and Western blotting, SV7 tert cells or control cells (DTC-1) were lysed and extracts were immunoprecipitated with C5 monoclonal antibody against MITF as described.18
Analysis of Active Mitogen-Activated Protein Kinase (MAPK) and TSC Protein Expression in SV7 Cells
SV7 cells were analyzed for expression of hamartin, tuberin, and active (phosphorylated) MAPK before introduction of telomerase. To determine whether tuberin, hamartin, and phosphorylated MAPK were appropriately expressed in our cells, proteins were extracted using MAPK lysis buffer (20 mmol/L Tris, pH 7.5, 10 mmol/L EGTA, 40 mmol/L ß-glycerophosphate, 1% Nonidet P-40, 2.5 mmol/L magnesium chloride, 2 mmol/L sodium orthovanadate, 1% aprotinin, 1% benzamidine). Equal amounts of protein, as determined using the bicinchoninic acid protocol (Pierce, Rockford, IL) were loaded on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels, using a standard SDS-polyacrylamide gel electrophoresis protocol. After electrophoresis, the gels were transferred to Immobilon-P membranes and probed with a phospho-specific MAPK antibody (New England Biolabs, Beverly, MA), a rabbit polyclonal anti-tuberin antibody (C20; Transduction Laboratory), or a rabbit polyclonal anti-hamartin antibody19 and developed using horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (ECL; Amersham, Arlington Heights, IL). Equal loading of samples was checked by stripping the blots and re-probing with an antibody to tubulin (clone DM1A; Sigma Chemical Co., St. Louis, MO). C20/A4 cells is a chondrocyte cell line derived by transfection of the pMKSVori- plasmid into primary human chondrocytes, and was used as a control cell line for SV40 expression.20
Co-Immunoprecipitation of Tuberin and Hamartin in SV7 Tert Cells
Cells were lysed in 20 mmol/L HEPES, 150 mmol/L NaCl, 1% Triton X-100, 10% glycerol, 1 mmol/L ethylenediaminetetraacetic acid, 10 mmol/L ß-mercaptoethanol, 0.5 mg/ml Pefabloc SC and cleared by centrifugation at 14,000 rpm for 10 minutes. The supernatants were precleared with Protein A/G agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) and immunoprecipitated with anti-Tsc 1 rabbit antibodies H2 or H4 (1 µg/ml) and anti Tsc 2 rabbit antibodies: tuberin (C-20) or tuberin (N-19). Prebled serum and purified rabbit IgG (Santa Cruz Biotechnology) served as a control. The immunocomplexes were pulled down with Protein A/G beads and washed three times with Tris-buffered saline buffer. The immunoprecipitates were resolved on SDS/polyacrylamide gel electrophoresis, immunoblotted for TSC1 with HM4 (1:1000) or TSC2 with C-20 or N-19, and detected with ECL (SuperSignal; West Pico, Pierce).
Genetic Analysis of Hamartin and Tuberin in SV7 Tert Cells
DNA was extracted from SV7 tert cells and analyzed according to the procedure of Au and colleagues.21 Primer pairs were developed to flank each exon of hamartin and tuberin, and amplification was accomplished by PCR. PCR products were electrophoresed at 2 W at 4°C, followed by gel-drying and autoradiography. Bands were examined by single strand conformation polymorphism analysis.
Analysis of akt Phosphorylation and PPAR Expression in
Angiomyolipoma Cells
Phosphoinositol-3-kinase (PI3K) is a major signal transduction
pathway involved in lipogenesis, angiogenesis, and tumorigenesis, and
is required for PPAR-mediated accentuation of lipogenic
differentiation.22-24
Thus, we examined SV7 tert cells
for expression of phosphorylated akt, a major downstream target of
phosphoinositol-3-kinase activation, and expression of PPAR. After
treatment, the cells were harvested at 5 x
107/ml in lysis buffer (0.05 mmol/L phosphate
buffer, pH 7.4, 5% glycerol, 1 mm ethylenediaminetetraacetic acid) and
sonicated briefly to break the cells. Equivalent amounts of protein
extraction (10 µl) were boiled with 10 µl 2x sample buffer
(Bio-Rad, Richmond, CA) and loaded on 10% SDS-polyacrylamide gels.
After electrophoresis, The gels were transferred to polyvinylidene
difluoride membrane at 450 mAmp for 2 hours in transfer buffer (25
mmol/L Tris,190 mmol/L glycine, 20% methanol, and 0.05% SDS). The
membranes were blocked in block buffer (10 mm Tris, pH 7.5, 0.1%
Tween-20, 100 mm NaCl, 5% nonfat dry milk) and probed with a
phospho-akt antibody,1:1000, (New England BioLabs, Beverly, MA) or
PPAP (E-8) antibody, 1:500, (Santa Cruz Biotechnology). After an
extensive wash in Tris plus Tween-20, the membranes were probed with
horseradish peroxidase-conjugated secondary antibody and detected by
chemiluminescence (Amersham).
Immunohistochemistry for PPAR in Angiomyolipoma Tissue
Five-µm sections of formalin-fixed, paraffin-embedded tissue
from the patient’s angiomyolipoma were tested for the presence of
PPAR using anti-PPAR antibodies (E-8, Santa Cruz Biotechnology)
at a concentration of 1:10 using an avidin-biotin complex technique and
steam heat-induced antigen retrieval. An avidin-biotinylated enzyme
complex kit (DAKO LSAB2, DAKO Corp.) was used in combination with the
automated DAKO Autostainer. Hematoxylin is used as a counterstain.
Negative controls were primary antibody replaced by buffer.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Primary cells were transfected with a plasmid encoding the entire
SV40 genome, except the origin of replication, into cells derived from
a spontaneous angiomyolipoma (Figure 1)
. Approximately 1 month after
transfection, foci of cells were noted, and seven foci were expanded.
Four of these clones rapidly underwent senescence, and were not studied
further. Three clones were examined by electron
microscopy.
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To further characterize the cells that were expanded from
the angiomyolipoma, electron microscopy was performed. Two populations
of cells were identified (Figure 2)
. The
first population demonstrated a fibroblastic appearance and lack of
abundant lysosomes (clone SV3, Figure 2A
). The second population
demonstrated an epithelioid morphology and abundant secondary
lysosomes, characteristic of authentic angiomyolipoma tissue (clones
SV6, SV7; Figure 2, B and C
).25
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Given that the SV7 cell line exhibited ultrastructural features of authentic angiomyolipoma tissue, we chose it for further study. To circumvent senescence and obtain a stable population of angiomyolipoma cells, we introduced telomerase into early passage SV7 cells, and obtained cells we named SV7 tert cells.
Immunohistochemical Analysis of SV7 Tert Cytospins
SV7 tert cells exhibited epithelioid features by routine
hematoxylin and eosin staining (Figure 3A)
and demonstrated nuclear staining
with MITF, a melanocytic transcription factor previously demonstrated
to be expressed in angiomyolipomas (Figure 3B)
.26
In
addition, cells were focally positive for cytoplasmic
staining with smooth muscle actin (Figure 3C)
, consistent with the
melanocytic and smooth muscle phenotype observed in angiomyolipomas.
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RNA and protein expression of MITF was examined by reverse
transcriptase-PCR using primers specific for the M and A isoforms of
MITF. Abundant expression of both M and A isoforms were observed in
control cells, and abundant A isoform was expressed in SV7 tert cells
(Figure 4A)
. MITF is also expressed on
the protein level in SV7 tert cells, with a higher molecular weight A
isoform band. This is consistent with the reverse transcriptase-PCR
analysis that shows predominant expression of the A isoform of MITF
(Figure 4B)
.
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SV7 tert genomic DNA was analyzed for mutations in Tsc
1 and Tsc 2 according to the methods of Au and
colleagues.18
No mutations in either of these genes were
observed (data not shown). Demonstration of physical interaction by
co-immunoprecipitation of tuberin and hamartin indicates that these
genes were functional in SV7 tert cells (data not shown). All cells
analyzed demonstrated expression of hamartin and tuberin (Figure 5)
. These proteins are the same size as
native tuberin and hamartin. In addition, SV7, but not control C20/A4
cells, demonstrated activation of MAP kinase signaling through
increased expression of phosphorylated (active) MAP kinase (Figure 5)
.
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Expression in SV7 Tert
Cells
In addition to MAPK activation, we wished to determine whether the
PI3K signal transduction pathway was activated.22,27
A
primary activity of activation of the phosphoinositol-3-kinase pathway
is phosphorylation of akt, a kinase involved in regulation of
apoptosis. High levels of phosphorylated akt were observed, and these
levels were down-regulated by the PI3K inhibitor LY294002, indicating
that phosphorylation of akt in these cells is dependent on PI3K
activity (Figure 6A)
.
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is a molecule implicated in lipocytic differentiation, and
activators of PPAR have been demonstrated to have therapeutic
activity in liposarcoma.28
SV7 tert cells demonstrated
expression of PPAR (Figure 6B)
, suggesting that this gene may be
involved in the lipomatous differentiation of angiomyolipomas.
Immunohistochemistry of the spontaneous angiomyolipoma from which SV7
tert cells were derived shows abundant staining of cells lining the
large diameter vessels (Figure 7)
.
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Five million cells were injected into 2-month-old male nude mice, in the presence or absence of Matrigel (Collaborative Bioscience, Waltham, MA). No tumors were observed after 4 months of observation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The precise cell of origin of angiomyolipomas is not identified, although they are speculated to arise from perivascular epithelioid cells (PEC cells).2,6 Angiomyolipoma is a member of a group of neoplasms that co-express melanocytic and smooth muscle markers, including lymphangiomyomatosis of the lung, and clear-cell sugar tumors.30,31 They are most commonly observed in tuberous sclerosis complex, but may occur sporadically, especially in the case of renal angiomyolipomas.32,33 This study is the first to describe the use of telomerase in obtaining distinct populations from a benign neoplasm, and will facilitate biochemical and genetic analysis of benign neoplasms, which are composed of admixtures of fibroblasts and neoplastic cells.
Angiomyolipomas have previously been shown to express markers of melanocytic differentiation, including the transcription factor MITF.26 MITF has been shown to exist in at least four isoforms, differing in the 5' end. MITF M is melanocytic-specific, whereas MITF A, the isoform expressed in our SV7 tert cells, is highly expressed physiologically in retinal pigment epithelium.34 Both MITF M and A isoforms potently transactivate melanocytic genes such as tyrosinase, and mice deficient in MITF M are white-colored with dark eyes.35 The ocular pigmentation is because of MITF A activity.36 We thus demonstrate that SV7 tert cells express MITF A mRNA and protein, similarly to MITF A expression in retinal pigment epithelium and osteoclasts.37,38
Telomerase has been extremely useful in obtaining stable populations of primary cells.39-41 Mesenchymal cells rapidly immortalize on overexpression of telomerase, whereas epithelial cells may require additional events, such as inactivation of p16.42,43 Remarkably, in vitro generation of human tumor cells requires introduction of other oncogenes, such as SV40 large T antigen and oncogenic H-ras, in addition to telomerase.44 The relative genetic stability of cells transduced with telomerase may allow in vivo reconstitution of organs with cells expanded from a patient, thus reducing the need for allogenic organ transplantation.45-47
In a survey of renal neoplasms, telomerase activity was detected in renal cell carcinoma, but not in angiomyolipoma or oncocytoma.48 A second case report of a hepatic angiomyolipoma detected no evidence of telomerase expression.49 Telomerase expression may be a late event in the rare transformation of benign angiomyolipomas to malignant PEComas. The lack of expression of telomerase in benign neoplasms such as angiomyolipoma may explain in part the difficulty in obtaining stable populations of cells from angiomyolipomas.
The availability of angiomyolipoma cell lines will be useful in elucidating biochemical and genetic aberrations that underlie this usually benign but clinically morbid tumor. In addition, we demonstrate the general utility of telomerase in obtaining and expanding populations of cells from benign neoplasms that should facilitate genetic and biochemical analysis of other benign tumors. Telomerase has been used to obtain stable populations of normal cells with preservation of phenotypic characteristics.42,50,51 We extend these findings to benign neoplasms. Currently, analysis of benign neoplasms is hindered by admixture of tumor stromal cells that may confound biochemical and genetic analysis.52 Knowledge of the signal transduction abnormalities in angiomyolipoma may result in novel medical therapy for these tumors, both in tuberous sclerosis and in spontaneous angiomyolipomas.
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Acknowledgements |
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Footnotes |
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Supported by grants from the National Tuberous Sclerosis Association (to J. L. A., R. Y., H. O., H. N., and D. G.), the American Skin Association (to J. L. A.), Emory Skin Disease Research Core Center grant P30AR42687, and National Institutes of Health grant AR02030.
Accepted for publication April 26, 2001.
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