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Blood, Vol. 94 No. 8 (October 15), 1999:
pp. 2854-2861
A Deletion in the Gene for Transforming Growth Factor  Type I
Receptor Abolishes Growth Regulation by Transforming Growth Factor
in a Cutaneous T-Cell Lymphoma
By
William P. Schiemann,
Walther M. Pfeifer,
Edi Levi,
Marshall E. Kadin, and
Harvey F. Lodish
From the Whitehead Institute for Biomedical Research, Cambridge; the
Department of Pathology, Beth Israel Deaconess Medical Center and
Harvard Medical School, Boston; and the Department of Biology,
Massachusetts Institute of Technology, Cambridge, MA.
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ABSTRACT |
Spontaneous regression of skin lesions is characteristic of
lymphomatoid papulosis (LyP), a clonal cutaneous lymphoproliferative disorder. A minority of LyP patients progress to anaplastic large cell
lymphoma (ALCL) in which skin lesions no longer regress and extracutaneous dissemination often occurs. In 1 such case, we developed
a tumor cell line, JK cells, and show that these cells are resistant to
the growth inhibitory effects of transforming growth factor  (TGF-) due to the loss of cell surface expression of the TGF-
type I receptor (TR-I). Reverse transcriptase-polymerase chain
reaction (RT-PCR) and sequencing of JK cell TR-I cDNA clones identified a deletion that spanned the last 178 bp of exon 1, including
the initiating methionine. Hybridization of a radiolabeled fragment
internal to the deletion was detected in the genomes of
TGF--responsive cells, but not in JK cells, indicating that they
contain no wild-type TR-I gene. PCR primers that flanked the deleted
TR-I region amplified a single band from JK cell genomic DNA that
lacked the last 178 bp of exon 1 and all of the  5 kb of intron 1. This JK cell-specific genomic TR-I PCR product was distinct from
products amplified from TGF--responsive cells and was also readily
detected in tumor biopsies obtained before the establishment of the JK
cell line. Our results identify the first inactivating mutation in
TR-I gene in a human lymphoma that renders it insensitive to growth
inhibition by TGF-.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
TRANSFORMING GROWTH factor- (TGF-)
is a multifunctional cytokine, which elicits diverse physiological
responses in numerous cell types, including hematopoietic cells,
hepatocytes, myoblasts, adipocytes, chrondrocytes, and
neurons.1-4 The initiation of transmembrane signaling and
the diverse biological activities of TGF- are mediated through the
combined actions of 2 distinct receptor Ser/Thr protein kinases, termed
the TGF- type I (TR-I) and type II (TR-II)
receptors.5-9 TGF--stimulated signal transduction commences following its binding to a homodimeric complex of TR-II, which then associates with, transphosphorylates, and stimulates the
protein kinase activity of a homodimeric complex of the
TR-I.4,10-13 Activated TR-I complexes then transduce
intracellular signals to the nucleus in part through the recruitment
and phosphorylation of either Smad2 or Smad3 within their C-terminal
Ser-Ser-X-Ser motif.14-18 Once activated, Smad2 or Smad3
associate with the shared signaling molecule Smad414,19,20;
the resulting complex translocates to the nucleus and works in concert
with additional transcription factors21-24 to alter the
transcription of a large repertoire of genes involved in regulating a
variety of biological responses, including differentiation, apoptosis,
and extracellular matrix production.2-4,11 Importantly,
TGF- also inhibits the proliferation of epithelial cells by
suppressing the expression of c-myc, cyclin A, and CDK4 and by
inducing the expression of the CDK inhibitors p15 and p21; together
these events lead to the hypophosphorylation of the retinoblastoma gene
product, Rb, and the sequestration of the transcription factor,
E2F-1.25,26
Cellular insensitivity to growth inhibition by TGF- is a hallmark in
the genesis and progression of human cancer, and in many instances, can
be linked directly to inactivating mutations in or the loss of
expression of various signaling molecules whose activities are
regulated by TGF-. For instance, mutations that inactivate Smad2
have been identified in human colorectal and lung
cancers,15,27 while those leading to the inactivation or
loss of expression of Smad4 have been found in human pancreatic, breast, colorectal, lung, ovarian, and head and neck
cancers.14,27,28 Although not yet identified as a cause of
human cancers,29,30 the gene for Smad3 functions as a tumor
suppressor in mice. In particular, targeted disruption of Smad3 leads
to the formation of colorectal adenocarcinomas capable of penetrating
the intestinal wall and metastasizing to distant
locations,31 suggesting that the gene for Smad3, like those
for Smads 2 and 4, is a tumor suppressor.
The receptors for TGF- also appear to function as tumor suppressors
in vivo. Mutations in or loss of the gene for the TR-II have been
described in a variety of human malignancies, including colon, gastric,
prostate, and retinal cancers, and in some T-cell lymphomas.32-39 With respect to TR-I, several reports
show a strong correlation between diminished cellular responsiveness to
TGF- and decreased expression of this receptor in B-cell chronic
lymphocytic leukemia, colon, and pancreatic cancers.40-43
Additionally, unknown genetic alterations in the TR-I gene have also
been reported as the cause of insensitivity to TGF- in prostate
cancer cells.44
Here we show that JK cells, a human CD30+ anaplastic large
cell lymphoma (ALCL) cell line derived from and clonally related to
lymphomatoid papulosis (LyP), are resistant to the growth inhibitory effects of TGF-. Furthermore, we show that the inability of JK cells
to respond to TGF- is due to a 178-bp deletion in exon 1 of the
TR-I gene, resulting in its loss of expression. Moreover, our
results have established that the TR-I gene, like those for Smad2,
Smad4, and TR-II, is a human tumor suppressor. Our results are the
first to identify a specific mutation in the TR-I gene leading to
the loss of its expression and consequently the loss of its
antitumorigenic qualities in a human T-cell lymphoma cell line.
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MATERIALS AND METHODS |
Patient history and characteristics of tumor cells in culture.
In 1987, a 49-year old male developed LyP, a cutaneous
lymphoproliferative disorder characterized by spontaneous regression of
skin lesions. The patient's lesions continued to wax and wane until
1996 when several tumor nodules developed on the right forearm, sacrum,
and the left buttock. The lesions on the right forearm and buttock
responded to local irradiation. A progressively enlarging ALCL lesion
of the buttock was biopsied and xenografted into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice,
and the resulting explanted SCID mouse tumor used to establish the population of cells used herein, termed JK cells. A complete
description of JK cells in culture will be described elsewhere
(manuscript in preparation). Cultured JK cells maintained
the anaplastic morphology and phenotype (CD30+,
CD15 , CD3+) of the original tumor, while
cytogenetics showed an aneuploid karyotype with marker chromosomes,
which were present 3 years earlier in a T-cell clone isolated from a
prior skin lesion of the patient's right arm. Studies of T-cell
receptor gene rearrangement showed that JK cells were clonally related
to all prior LyP skin lesions analyzed dating back to
1988.45
[3H]Thymidine incorporation assay.
Mv1Lu or JK cells were plated onto 6-well plates at a density of
100,000 cells/well and were incubated in the absence or presence of
increasing concentrations of TGF-1 (0 to 20 ng/mL) for 48 hours at
37°C. During the final 4 hours of agonist treatment, cellular DNA
was radiolabeled by inclusion of [3H]thymidine to the
culture medium. Upon completion of agonist stimulation, the cells were
harvested in phosphate-buffered saline (PBS) supplemented with 10 mmol/L EDTA and 10% fetal bovine serum before their isolation and
concentration by centrifugation at 1,500 rpm for 3 minutes at room
temperature. Quantitation of [3H]thymidine incorporation
was measured using the SPA [3H]thymidine uptake assay
system (Amersham, Arlington Heights, IL) with slight
modification. Briefly, cell pellets were resuspended in 130 µL of
PBS, transferred to scintillation vials containing 75 µL bead/lysis
buffer mixture, and vortexed for 2 minutes at room temperature on a
multitube vortexer (VWR, setting 2). Enhancer (25 µL) was added to
each vial, and the samples were vortexed for 2 minutes immediately
before scintillation counting in a Beckman LS6500 liquid scintillation
counter (Beckman Coulter, Fullerton, CA).
Recombinant glutathione S-transferase (GST)-Smad3
phosphorylation assay.
Quiescent Mv1Lu, human HT1080 fibrosarcoma, human MDA-MB-231 breast
cancer or JK cells were incubated in the absence or presence of 20 ng/mL TGF-1 for 10 minutes at 37°C. Cytokine stimulations were
terminated by washing the cells 2 times in ice-cold PBS, and cells were
immediately lysed in harvesting buffer (50 mmol/L -glycerophosphate,
150 mmol/L NaCl, 1.5 mmol/L EGTA, 1 mmol/L dithiothreitol
(DTT), 0.1 mmol/L sodium vanadate, 1 mmol/L benzamidine, and 10 µg/mL leupeptin, pH 7.3) supplemented with 1% Triton X-100. Cell extracts were solubilized on ice for 60 minutes and were subsequently clarified by centrifugation for 10 minutes at 4°C. Immunoprecipitation of TGF- receptor complexes was accomplished by
rotating the clarified cell extracts (~ 1.5 mg
protein/tube) in the presence of anti-TR-II antibody and protein
A-sepharose for ~2 hours at 4°C. The resulting immunocomplexes
were recovered by brief centrifugation and were subsequently subjected
to 2 washes in lysis buffer, followed by 2 washes in harvesting buffer
lacking NaCl. TGF- receptor phosphotransferase activity against
recombinant GST-Smad3 was measured for 30 minutes at 30°C in a
final reaction mixture of 40 µL consisting of 30 µL of TGF-
receptor immunocomplexes, 25 mmol/L -glycerophosphate (pH 7.3), 0.5 mmol/L DTT, 1.25 mmol/L EGTA, 50 µmol/L sodium vanadate, 10 mmol/L
MgCl2, and 100 µmol/L adenosine triphosphate
(ATP) ([ -32P]ATP, 2,000 cpm/pmol),
and 5 µg of recombinant GST-Smad3. Protein kinase reactions were
quenched by addition of 13.5 µL of 4× Laemmli sample buffer and
were subsequently boiled for 5 minutes before fractionation through
10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE). TGF-1-stimulated phosphorylation of recombinant
GST-Smad3 was detected by autoradiography of the dried gels.
[125I]TGF-1 radioligand binding and
cross-linking assay.
Radioligand binding and cross-linking of iodinated TGF-1 to Mv1Lu
or JK cells (5 × 106 cells/tube) and the subsequent
immunoprecipitation of cytokine/receptor complexes with anti-TR-I
and -TR-II antibodies was performed as described
previously.40 TGF-1 bound to cell surface TR-I and
TR-II was visualized by autoradiography of the dried gels.
Northern blot analysis.
Total RNA was isolated from Mv1Lu or JK cells using the RNAzol B
reagent (Tel-Test, Inc, Friendswood, TX) according to the manufacturer's recommendations. Five micrograms of total RNA was fractionated through 1% agarose/formaldehyde gels, immobilized to
nylon membrane, and subsequently incubated at 68°C for 80 minutes in ExpressHyb hybridization solution (Clontech, Palo Alto,
CA) containing a 32P-radiolabeled probe
encoding to nucleotides 226-1536 of the TR-I cDNA. After
hybridization, the membrane was washed for 40 minutes at room
temperature in 2× SSC/0.05% SDS, followed by 80 minutes of
washing at 50°C in 0.1× SSC/0.1% SDS before visualization of the TR-I mRNA by autoradiography.
cDNA synthesis and PCR of JK cell TR-I clones.
To synthesize JK cell TR-I cDNAs, 800 ng of JK cell total RNA was
primed with oligo-dT and reverse transcribed using Superscript II
(GIBCO-BRL, Rockville, MD) according to the
manufacturer's recommendations. A total of 2 µL of resulting cDNA
mixture was used in 50 µL PCR reactions using the Elongase enzyme
mixture (GIBCO-BRL) in the presence of 10% dimethyl sulfoxide (DMSO)
to amplify full-length JK cell TR-I cDNA. The 5' and 3'
oligonucleotides were engineered to contain Spe I or Hind III
restriction sites, respectively, to facilitate subsequent subcloning of
amplified products into pBluescript. The sequences of the human TR-I
primers used were: (1) 5' oligo;
5'-ACTAGTACTAGTGGACGCGCGTCCTCCGAGCAG, corresponding to positions
 141 to 121 relative to the initiating ATG, and (2)
3' oligo; 5'-AAGCTTAAGCTTGAGAGTTCAGGCAAAGCTGTA,
corresponding to positions 1516-1536. After an initial denaturation
step for 2 minutes at 94°C, the PCR amplification reaction
conditions proceeded through 30 seconds at 94°C, 30 seconds at
55°C, and 2.5 minutes at 68°C for 40 cycles, followed by a
final extension for 5 minutes at 68°C. The resulting ~1.6 kb PCR
fragments were subcloned into the Spe I/Hind III sites of pBluescript,
propagated in Escherichia coli, and sequenced in their entirety
across both strands on an Applied Biosystems 377A DNA sequencing system
(Perkin Elmer, Applied Biosystems, Foster City, CA).
Genomic PCR.
Genomic DNA from TGF--responsive cells or JK cells was isolated
using the QIAamp Blood Kit (Qiagen, Valencia, CA) according to the
manufacturer's recommendations. Amplification of exon 1 of TR-I
gene from TGF--responsive cells or JK cells was accomplished using
the Advantage-GC genomic polymerase mix (Clontech) with the flanking
intronic primer pair as described previously.46 The
resulting PCR products were isolated from unincorporated nucleotides and primers before separation through a 2.5% agarose/TAE gel.
Additionally, 1 µg of genomic DNA was subjected to PCR amplification
in 50-µL reaction volumes using the Elongase enzyme mixture in the
presence of 10% DMSO and primers that flanked the 178-bp deletion
identified in JK cell TR-I cDNA. The sequences of the human TR-I
primers used were: (1) 5' oligo;
5'-ACTAGTACTAGTGGACGCGCGTCCTCCGAGCAG, corresponding to positions
141 to 121 relative to the initiating methionine, and (2)
3' oligo; 5'-ATATGTTGTAGTCACAGACCCAGT, corresponding to
positions 274 to 297 relative to the initiating ATG. After an initial
denaturation step for 2 minutes at 94°C, the PCR amplification reaction conditions proceeded through 30 seconds at 94°C, 30 seconds at 50°C, and 60 seconds at 68°C for 30 cycles, which
was followed by a final extension for 5 minutes at 68°C. The
resulting PCR products were isolated from unincorporated nucleotides
and primers before separation through a 2.5% agarose/TAE gel. The
products of interest were excised from the gel, purified using Qiaex II beads (Qiagen) according to recommended protocols, and subsequently sequenced in their entirety across both strands on an Applied Biosystems 377A DNA sequencing system.
Patient cutaneous tumor lesions biopsied in 1989 and 1996 were obtained
from the Beth Israel Deaconess Hospital Department of Pathology
archives. Genomic DNA from frozen tumor tissue (1989) was isolated
using DNAzol (GIBCO-BRL) according to the manufacturer's recommendations. Paraffin-embedded material (1996) was microdissected using a 30 gauge needle to scrape a 5-µm thick, Eosin-stained tumor
sample. Tumor scrapings were placed in extraction buffer (50 mmol/L
Tris, 1 mmol/L EDTA, and 0.5% Tween-20) and incubated in the presence
of proteinase K for 48 hours at 37°C. Upon completion of proteinase
K digestion, 3 µL of heat inactivated sample was subjected to PCR
amplification in 25-µL reaction volumes using the Ready to Go PCR
Beads (Pharmacia Biotech, Piscataway, NJ) and primers
that flanked the 178-bp deletion identified in JK cell TR-I cDNA.
The sequences of the human TR-I primers used were: (1) 5'
oligo; 5'-CCTCCGAGCAGTTACAAAGG, corresponding to positions
131 to 112 relative to the initiating methionine, and (2)
3' oligo; 5'-AACGGCCTATCTCGAGGAAT, corresponding to
positions 232 to 251 relative to the initiating ATG. After an initial
denaturation step for 7 minutes at 94°C, the PCR amplification
reaction conditions proceeded through 50 seconds at 94°C, 50 seconds at 60°C, and 90 seconds at 72°C for 35 cycles, which
was followed by a final extension for 7 minutes at 72°C. The
resulting PCR products were fractionated through a 2% agarose/TAE gel.
Southern blotting.
A total of 10 µg of genomic DNA was digested overnight at 37°C
with Hind III and subsequently transferred to a nylon membrane. Immobilized DNA was hybridized overnight at 65°C in modified
Church-Gilbert hybridization solution (500 mmol/L NaHPO4,
7% SDS, 1 mmol/L EDTA, and 1% bovine serum albumin [BSA], pH 7.2)
containing a 32P-radiolabeled probe corresponding to the
178-bp sequence absent in JK cell TR-I cDNA (ie, bases 81 to
96 relative to the initiating ATG). Upon completion of hybridization,
the membrane was washed at 65°C in several changes of
Church-Gilbert wash solution (40 mmol/L NaHPO4, 1 mmol/L
EDTA, and 1% SDS, pH 7.2) before visualization of the TR-I gene by autoradiography.
| |
RESULTS |
Because we had previously shown that TGF--mediated growth
regulation can be impaired in cells that have undergone progression from LyP to ALCL,37,38 we sought to determine whether JK
cells, which were derived from an ALCL lesion, remained competent to undergo growth arrest as determined by a [3H]thymidine
incorporation assay in response to TGF- administration. In contrast
to the TGF--mediated response measured in Mv1Lu cells (Fig 1) and the expected growth inhibition
of normal T-lymphocytes,47-52 incubation of JK cells in the
presence of increasing concentrations of TGF- failed to inhibit
their ability to synthesize DNA (Fig 1).
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| Fig 1.
JK cells fail to undergo growth arrest in response to
TGF-. Mv1Lu ( ) or JK ( ) cells were incubated in the absence or
presence of increasing concentrations of TGF-1 (0 to 20 ng/mL) for
48 hours at 37°C. During the final 4 hours of agonist treatment,
cellular DNA was radiolabeled by addition of
[3H]thymidine in the culture medium. Upon completion of
agonist stimulations, the cells were harvested and prepared for
scintillation counting to determine radionucleotide incorporation into
cellular DNA as described in Materials and Methods. Data are the mean
(± standard error of mean [SEM]) of 3 independent experiments
normalized to untreated controls.
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Defects in or loss of expression of the TR-II for TGF- has been
linked to the growth of cutaneous T-cell lymphoma.37-39 As a direct measure of JK cell TGF- receptor functioning, we performed an in vitro protein kinase assay that assessed the ability of immunoprecipitated TGF- receptors to phosphorylate exogenously added
GST-Smad3 fusion protein. Unlike agonist-stimulated TGF- receptors
isolated from TGF--responsive cells (ie, Mv1Lu, HT1080 fibrosarcoma,
or MDA-MB-231 breast carcinoma cells), immunocomplexes of JK cell
TGF- receptors failed to phosphorylate recombinant GST-Smad3 in an
agonist-dependent manner (Fig 2A). Indeed,
the inability of activated JK cell TGF- receptors to phosphorylate GST-Smad3 is consistent with the refractoriness of JK cells to TGF-
administration and suggests that the abnormal responses of JK cells to
TGF- treatment may arise from alterations in either, or both, of the
genes for the TGF- receptors.
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| Fig 2.
Analysis of JK cell TR-I receptors. (A)
Immunocomplexes of JK cell TR-I fail to phosphorylate recombinant
GST-Smad3. Mv1Lu (Mv1), human HT1080 fibrosarcoma (HT), human
MDA-MB-231 breast cancer (231), or JK cells were incubated in the
absence or presence of TGF-1 for 10 minutes at 37°C. Triton
X-100 solubilized cell extracts were prepared and subjected to
immunoprecipitation with anti-TGF- type II receptor antibodies, and
the resulting immunocomplexes were incubated in the presence of
recombinant GST-Smad3 and [-32P]ATP as described in
Materials and Methods. Data shown is a representative autoradiograph
depicting TGF--stimulated phosphorylation of recombinant GST-Smad3.
(B) Loss of cell surface expression of TR-I receptors in JK cells.
Mv1Lu or JK cells (5 × 106 cells/tube) were
incubated in the presence of 250 pmol/L [125I]TGF-1
for 90 minutes at 4°C before addition of disuccinimidyl suberate to
cross-link the cytokine/receptor complexes. Triton X-100 solubilized
cell extracts were prepared and subjected to immunoprecipitation with
anti-TR-I or -TR-II receptor antibodies as described in
Materials and Methods. Data shown is a representative autoradiograph
depicting iodinated TGF-1 bound to its TR-I and TR-II
receptors as indicated. (C) TR-I mRNA is expressed normally in JK
cells. A total of 5 µg of total mRNA was hybridized to a
32P-radiolabeled probe corresponding to nucleotides
226-1536 of the TR-I cDNA. Data shown is a representative
autoradiograph of the ~6.5 kb TR-I mRNA transcript in Mv1Lu and JK
cells.
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To test this hypothesis, we performed a radioligand binding and
cross-linking experiment that measured the ability of iodinated TGF-
to form complexes with its receptor polypeptides. As expected, addition
of iodinated TGF-1 to Mv1Lu cells facilitated the formation of
cytokine/receptor complexes comprised of the 2 signaling receptor polypeptides for TGF-, TR-I, and TR-II (Fig 2B). In contrast to Mv1Lu cells and the expected TGF- receptor binding profiles of
normal T-lymphocytes,37,47,50-52 incubation of iodinated
TGF-1 with JK cells showed that the TR-II was expressed normally
to the cell surface, while expression of TR-I capable of binding TGF- was completely absent from the surface of JK cells (Fig 2B).
This finding suggests that the insensitivity of JK cells to TGF- may
be due to defects in the gene for TR-I.
We next subjected JK cell total RNA to Northern blot analysis using a
32P-radiolabeled probe that encoded nucleotides 226-1536 of
the human TR-I. As shown in Fig 2C, a single TR-I transcript of ~6.5 kb was detected in the TGF--responsive Mv1Lu cells, as well as in the TGF--unresponsive JK cells. This result indicates that the loss of cell surface TR-I expression in JK cells cannot be due
to deletion of the gene for TR-I or in its ability to be transcribed, but may instead be due to the presence of a mutation(s) within its coding sequence.
To test this hypothesis, we performed RT-PCR on JK cell total RNA to
generate full-length TR-I cDNA clones. Sequencing the complete
coding region of individual JK cell TR-I cDNA clones identified a
178-bp deletion in 12 of 13 clones. As shown in
Fig 3, this deletion spanned the last 178 bp of exon 1 and eliminated the initiating methionine. No additional
mutations were found within the remaining coding sequence of JK cell
TR-I cDNA clones. Consistent with the phenotype of JK cells, ectopic
expression of JK TR-I cDNA in Cos-7 cells failed to produce a
protein capable of forming complexes with TR-II upon addition of
iodinated TGF-, nor did its expression alter or reconstitute
TGF--stimulated gene expression in Mv1Lu or R1B
cells,53 respectively (data not shown). The lone remaining
TR-I clone was wild-type both in its sequence and function when
ectopically expressed in mammalian cells (data not shown). It is
important to note that JK cells are a population, not a clone, of the
explanted SCID mouse tumor cells, and as such, it is likely that a
minute quantity of wild-type cells would be present and detected by
RT-PCR analysis, but would escape detection in assays designed to
measure TGF- function (Figs 1 and 2). Taken together, these results
indicate that the inability of JK cells to respond to TGF- results
from the deletion of the initiating methionine contained within the
deleted 178 bp of exon 1, and consequently prevents the translation of
the TR-I polypeptide.
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| Fig 3.
Intron-exon organization of the human TR-I:
identification and localization of an N-terminal deletion in JK cell
TR-I cDNA. JK cell total RNA was subjected to RT-PCR analysis to
facilitate the cloning and isolation of full-length JK cell TR-I
cDNAs as described in Materials and Methods. Shown is the
transcriptional start site (pos. 236) and the intron-exon
organization of the human TR-I as described recently by Vellucci and
Reiss.63 As depicted, the TR-I is comprised of 9 exons
spanning ~31 kb of genomic DNA sequence. Sequencing of 13 JK cell
TR-I cDNA clones generated by RT-PCR demonstrated the presence in 12 clones of a 178-bp deletion, beginning at position 81 relative to
the initiating methionine and concluding at position +96. This
deletion eliminates the initiating methionine and the final 178 bp of
exon 1 of the TR-I in JK cells.
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To exclude the possibility that the deletion identified in the TR-I
in JK cells was an artifact of PCR, we subjected JK cell genomic DNA to
PCR and Southern blot analyses (Fig
4A). First, use of flanking primers designed to amplify
exon 1 (primers 1 and 2, Fig 4A) of TR-I failed to elicit any signal
from JK cell genomic DNA, while the expected 255-bp fragment was
readily detected in genomic DNA of TGF--responsive cells (Fig 4B,
upper panel). The inability to detect a truncated TR-I exon 1 signal
from JK cell genomic DNA implied that the 178-bp deletion identified in its cDNA may encompass part, or all, of its first intron, but would not
alter the integrity of exon 2, which was wild-type in RT-PCR analyses
(Fig 3). Second, and consistent with this hypothesis, we found that
amplification of JK cell genomic DNA with primers that flanked the
deleted TR-I cDNA region produced a single, unique band of 260 bp
that was not evident in the genomes of TGF--responsive cells, which
exhibited an ~500 bp fragment (Fig 4B, lower panel). Sequencing of
this 260-bp fragment showed it to be identical to that found in its
TR-I cDNA, indicating that the deletion in the gene for the TR-I
in JK cells is ~5 kb, and encompasses the last 178 bp of exon 1 and
all of the first intron (Fig 3). Sequence analysis of the 500-bp PCR
product amplified from TGF--responsive cells failed to yield
TR-I sequence, and as such, represents a nonspecific PCR product.
Although we do not yet know why this nonspecific PCR product was
obtained exclusively from cells harboring wild-type TR-I genes, its
appearance solely in TGF--responsive cells may be due to the
presence of intron 1 in their TR-I genes, a region that is absent in
the TR-I genes of JK cells. For instance, the PCR reaction
conditions used in these experiments (ie, 60-second extension segments)
would not be expected to amplify intron 1 in its entirety, and as such,
the appearance of the nonspecific 500 bp product may arise from
premature termination of PCR product elongation or from decreased
fidelity of the polymerase due to the high GC-content and repetitive
sequences inherent to introns. JK cells, whose TR-I gene lacks
intron 1 (Fig 3), are not subject to the intronic effects of their
TGF--responding counterparts, and consequently fail to produce the
nonspecific 500 bp product that is consistently and exclusively found
in TGF--responsive cells.
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| Fig 4.
Genomic analysis of TR-I in JK cells. (A) Schematic
depicting the PCR primers and Southern blotting probe used to analyze
the intron-exon structure surrounding exons 1 and 2 of the TR-I
gene. Shown is exons 1 and 2 of TR-I (stippled boxes) and their
intervening intron. The hatched box of exon 1 represents the 178-bp
deletion identified by RT-PCR analysis of JK cell mRNA, and the thick
bar underlying this region depicts the 32P-radiolabeled PCR
fragment used in Southern blotting experiments. Amplification of exon 1 from genomic DNA was accomplished using primer pair 1 and 2 to amplify
the normal 255-bp product. Exon 1 and 2 initiated amplification across
intron 1 was accomplished using primer pair 3 and 4 or primer pair 5 and 6 on genomic DNA obtained from cultured cells or patient biopsies,
respectively. Primer sequences and PCR reaction conditions are
described in Materials and Methods. (B) PCR of genomic DNA identifies
an ~5 kb deletion in JK cell TR-I gene. (Upper panel) A total of
100 ng of genomic DNA from JK, HepG2 hepatoma (Hep), HT1080
fibrosarcoma (HT), or MDA-MB-231 breast cancer (231) cells was
subjected to PCR amplification using primer pair 1 and 2 that flanked
exon 1 of the human TR-I. The resulting PCR products were
fractionated through a 2.5% agarose/TAE gel as described in Materials
and Methods. Data shown is a representative picture of an ethidium
bromide-stained gel demonstrating the presence of the 255-bp TR-I
product only in TGF--responsive cells. (Lower panel) One microgram
of genomic DNA from JK, HepG2 (Hep), HT1080 (HT), or MDA-MB-231 (231)
cells was subjected to PCR amplification using primer pair 3 and 4, which flanked the 178-bp deletion in the JK cell TR-I cDNA. Shown is
a representative picture of an ethidium bromide-stained gel
demonstrating the presence only in JK cells of a single amplified PCR
product, whose size (260 bp) and sequence was identical to that of the
JK cell TR-I cDNA. (C) Radiolabeled deletion fragment fails to
hybridize with JK cell genomic DNA. Ten micrograms of genomic DNA from
JK, HepG2 (Hep), HT1080 (HT), or MDA-MB-231 (231) cells was digested
overnight at 37°C with Hind III, and after transfer to nylon
membrane, hybridized with a 32P-radiolabeled probe
corresponding to the 178-bp sequence absent in JK cell TR-I cDNA as
described in Materials and Methods. Data shown is a representative
autoradiograph demonstrating the presence of a hybridizing signal in
the genomes of TGF--responsive cells, but not in JK cells. (D)
Detection of mutated TR-I in tumor biopsies obtained before
establishment of the JK cell line. JK cell genomic DNA (Gen), cDNA, or
genomic DNA obtained from patient tumor biopsies performed in 1989 and
1996 were subjected to PCR amplification using primer pair 5 and 6, which flanked the 178-bp deletion in the JK cell TR-I cDNA. Shown is
a representative picture of an ethidium bromide-stained gel
demonstrating the presence of mutated TR-I gene in the patient's
primary tumor samples before the establishment of the JK cell line.
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Third, as shown in Fig 4C, hybridization of a
32P-radiolabeled probe encoding nucleotides 81 to 96 of human TR-I cDNA (ie, the sequence deleted in JK cell TR-I
cDNA) to a Southern blot of genomic DNA of TGF--responsive cells
elicited a strong hybridizing signal. Consistent with the results in
Fig 4B, hybridization of JK cell genomic DNA with this same
32P-radiolabeled probe was negative (Fig 4C). The absence
of wild-type bands both in the JK cell genomic PCR and JK Southern blot
indicates that the population of JK cells no longer contain any
significant amount of wild-type TR-I sequences within their genome.
Lastly, we asked whether the deletion in the JK cell TR-I gene was
present in the patient's primary tumor samples. Amplification of
patient genomic DNA obtained from tumor biopsies performed in 1989 and
1996 with primers that flanked the deleted region of JK cell TR-I
cDNA produced a product that was identical to that amplified from
either JK cell genomic DNA or cDNA (Fig 4D). This finding demonstrates
that the deletion in the JK cell TR-I gene did not arise as a
consequence of its derivation or time in culture, but rather was
present during the course of the patient's lymphoma.
| |
DISCUSSION |
Although between 10% and 20% of patients with LyP develop malignant
lymphoma,54 the genetic alterations underlying this progression are currently unknown. We previously described the inability of many lymphoma cells to undergo growth arrest in response to TGF- after their progression from LyP to malignant cutaneous tumors.37 In the present study, we sought to determine the
molecular basis responsible for resistance to TGF--mediated growth
inhibition in JK cells, a human cutaneous T-cell lymphoma cell line
derived from an advanced cutaneous ALCL. We identified an ~5 kb
deletion in the gene for the TR-I that eliminates the last 178 bp of
exon 1, including the initiating methionine, and all of intron 1. Consistent with the phenotype of JK cells, expression of mutated JK
cell TR-I cDNA in mammalian cells failed to produce a polypeptide capable of interacting with TR-II upon addition of TGF-, nor did
its expression reconstitute TGF--mediated signaling in R1B cells,
which lack TR-I (data not shown). Indeed, our results suggest that
the insensitivity of JK cells to TGF- arises from their inability to
mediate efficient translation of TR-I transcripts, and as such,
represents the first report to identify and describe a specific
mutation in the gene for the TR-I responsible for liberating cells
from the antiproliferative effects of TGF-.
With respect to involvement in the generation and progression of human
cancer, TGF- and its intracellular signaling proteins are widely
established tumor suppressors. For instance, the
TGF--stimulated signaling molecules Smad2 and
Smad4,14,15,27,28 and possibly Smad3,31 are
frequently mutated or deleted in human cancers. Similarly, inactivating
mutations in or loss of TR-II expression causes insensitivity to
TGF- in a variety of human neoplasms, including some colon, gastric,
prostate, and retinal cancers, and some T-cell
lymphomas.32-39
Interestingly, while a number of studies have clearly established the
importance of TR-I as the initiator of TGF--stimulated signaling
events after its activation by TR-II,5,10,55,56 few
studies have provided definitive evidence of its importance as a tumor
suppressor gene in humans. Indeed, Chen et
al46 have recently identified a S387Y substitution that
produces only a modest inhibition in TR-I-mediated signaling, but
is nonetheless highly associated with metastatic breast cancer.
Additionally, a common polymorphism localized within the signal
sequence of the TR-I gene of normal volunteers and patients with
acute myeloid leukemia has also been described recently.57
Although this polymorphism was detected at higher frequencies in
transformed versus nontransformed cells, a complete and thorough
understanding of its biological and clinical significance remains to be determined.
Insensitivity to TGF- has, however, been attributed to reduced or
lost expression of cell surface TR-I in colon cancer or B-cell
chronic lymphocytic leukemia,40,41 respectively. Diminished expression of TR-I has also been shown to mediate resistance of
pancreatic cancer cells to TGF-,42,43 while homozygous deletion of the TR-I gene has recently been found in 1% of all pancreatic cancers surveyed.58 Similarly, an unknown
genetic alteration leading to the loss of TR-I transcription was
capable of freeing prostate cancer cells from the growth inhibitory
effects of TGF-.44 Our results identifying a TR-I
mutation in a human lymphoma, together with the above findings,
indicate that the TR-I gene is genetically unstable and highly
susceptible to inactivating mutations capable of facilitating the
formation of malignant cells.
Although 1 in 13 JK cell TR-I cDNA clones sequenced after RT-PCR
analysis was wild-type both in its sequence and function, this clone
and its parental cell are not reflective of the population of JK cells
as a whole. Because JK cells are a population, not a clone, of the
explanted SCID mouse tumor cells, it is likely that the JK cell line
might harbor a small percentage of wild-type cells that escape
detection in TGF- functional assays (Figs 1 and 2), but are detected
in RT-PCR experiments. However, our inability to detect by Southern
blotting a hybridizing signal in JK cells with a probe internal to the
TR-I deletion in JK cell cDNA, together with the absence of multiple
wild-type products obtained from PCR of JK cell genomic DNA,
demonstrates that the vast majority of the population of JK cells no
longer harbor wild-type TR-I genes within their genome. These
results suggest that JK cells have undergone a loss of heterozygosity
at the TR-I locus, leaving only the deletion containing TR-I
gene. This hypothesis is in keeping with the paradigm of inactivation
of tumor suppressor genes (ie, inactivating mutation, followed by a
loss of heterozygosity), and with the recent finding that late in the
development of squamous cell carcinoma, 65% of all tumors exhibit a
loss of heterozygosity at chromosome position 9q22.3,59 a
region to which the gene for the TR-I has been
assigned.57
Although the exact role the JK cell TR-I deletion played during the
course of the patient's disease remains unknown, it is plausible that
the loss of TR-I expression mediated by the JK deletion after the
loss of heterozygosity at the TR-I locus conferred a selective
growth advantage to the developing tumor. Although we can detect
mutated TR-I early in the clinical course of the patient's
lymphoma, we cannot ascertain exactly when homozygosity at the TR-I
locus occurred; however, as the loss of TR-I heterozygosity occurred
before the establishment of the JK cell line, it is tempting to
speculate that this event may have taken place during the progression from LyP to ALCL. Studies of TGF-1-deficient mice showed that although approximately 50% of all pups died in utero, the remaining littermates developed normally and survived to term, only to succumb to
severe inflammatory responses within 3 to 4 weeks after
parturition.3,60,61 These findings suggest that TGF-
plays a vital role in suppressing immune cell activation and
proliferation in vivo. With respect to the patient's developing
lymphoma, the loss of TGF- sensitivity would have removed the
immunosuppressive properties of TGF- and enhanced cell
proliferation. Furthermore, because malignant cells typically
upregulate their production and secretion of TGF-, ancestral cells
homozygous for the JK TR-I deletion would also be expected to
experience increased protection against immune system surveillance by
inhibiting the clonal expansion of tumor-infiltrating lymphocytes.62 Clinically, the presence of TR-I, not
TR-II, was found to be a highly predictive marker of prostate cancer treatability and patient survival, with loss of TR-I expression resulting in higher Gleason scores (ie, more poorly differentiated), more advanced clinical stages, decreased survival rates, and increased serological recurrence after radical prostatectomy.34
Although studies in more patients are clearly needed, it is tempting to speculate that the integrity of TR-I in human lymphomas may be used
as a prognostic indicator for patient treatability and survival.
In summary, our results are the first to identify a specific mutation
in the TR-I gene leading not only to the loss of its expression, but
also to the loss of its tumor suppressive properties in human T-cell
lymphoma. More studies are clearly needed, and are currently ongoing,
to further our understanding of the role of the TR-I during the
development and progression of cancer, particularly those involving T-
and B-cell lymphomas, where alterations in receptor signaling appear to
play a role in the development of the malignant phenotype.
| |
ACKNOWLEDGMENT |
The authors thank Dr Christina Rhodes for help and suggestions with the
RT-PCR reactions, Dr Xeudong Liu for providing the GST-Smad3 construct,
and Dr Rebecca Wells for providing iodinated TGF-1 and anti-TR-I
and -TR-II antibodies. Members of the Lodish laboratory are
gratefully acknowledged for providing useful technical suggestions and
numerous helpful discussions. Drs Ralph Lin and Allen Sirotkin are
thanked for critical reading of the manuscript.
| |
FOOTNOTES |
Submitted March 10, 1999; accepted June 12, 1999.
Supported by Grant No. CA73161-01 from the National Cancer Institute
(to W.P.S.), Grant No. CA63260 from the National Institutes of Health
(to H.F.L.), by the Leukemia Society of America (M.E.K.), Grant No.
ROG-125-01 from the American Cancer Society (to M.E.K.), a grant from
Berlex, Inc (to M.E.K), and by the Deutsche Krebshilfe (W.M.P.).
Address requests for JK cell line to Marshall E. Kadin, MD, Department
of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical
School, Boston, MA 02215.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Harvey F. Lodish, PhD,
Whitehead Institute for Biomedical Research, Nine Cambridge Center,
Cambridge, MA 02142; e-mail: lodish{at}wi.mit.edu.
| |
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Human T-cell lymphotropic virus oncoprotein Tax represses TGF-beta 1 signaling in human T cells via c-Jun activation: a potential mechanism of HTLV-I leukemogenesis
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[Abstract]
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TOP
ABSTRACT
INTRODUCTION