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NEOPLASIA
From the Division of Hematology and Oncology,
University of Pennsylvania, Philadelphia, PA.
TEL/platelet-derived growth factor receptor Tyrosine kinase fusion proteins are the products of
a growing family of oncogenes associated with both solid tumors and
hematologic malignancies.1 The best known tyrosine kinase
fusion protein is the BCR/ABL tyrosine kinase that results from a
t(9;22) translocation in patients with chronic myeloid
leukemia.2 Previous work has demonstrated that BCR/ABL
activates multiple signal transduction pathways, including the
phosphatidylinositol-3 (PI3) kinase pathway and that transformation by
BCR/ABL requires activation of PI3 kinase.3-6 To determine
if tyrosine kinase fusion proteins share common mechanisms of
transformation or if each functions in unique ways, we have chosen to
study the TEL/platelet-derived growth factor receptor TEL/PDGF Several signaling pathways have been identified as being activated by
TEL/PDGF Here, we report that TEL/PDGF Reagents
Growth curves and cell cycle analysis
Western blotting Cells were washed once in phosphate-buffered saline and lysed in lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 1% Triton-X 100 plus protease and phosphatase inhibitors). Lysates were clarified by centrifugation and protein quantitated by using a modified Bradford reagent (Bio-Rad Laboratories, Hercules, CA). Lysates (100 µg) were loaded on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), proteins were separated by electrophoresis and blotted onto nitrocellulose or polyvinylidene diflouride. Blots were blocked in 5% dry milk in TBST (0.1% Tween-20, 0.01 M Tris-HCl, pH 7.6, 150 mM NaCl) or 5% bovine serum albumin (Sigma), rinsed in TBST for 5 seconds and incubated for 2 hours at room temperature in primary antibody. Blots were washed and incubated with horseradish peroxidase-conjugated secondary antibody and developed with enhanced chemiluminescence according to the manufacturer's directions (Amersham Biosciences, Piscataway, NJ). Antibodies used were as follows: phospho-Akt and phospho p70S6 kinase, Akt, p70S6 kinase (New England Biolabs, Beverly, MA), p21, p27, cdk4, and Cyclin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA).Immune complex protein kinase assay Pellets from cells were lysed with lysis buffer; 10 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton-X 100, 0.1% SDS, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 1 µg/mL pepstatin, 5 mM sodium orthovanadate, 50 mM NaF, 50 mM Na-pyrophosphate, and 150 µM phenylmethylsulfonyl fluoride. Total protein concentration in each sample was determined by using a micro BCA method (Bio-Rad) according to the manufacturer's instructions. We then transferred a 100-µg cell extract to Microfuge tube (total volume in 500 µL IP buffer). Immunoprecipitations were carried out by incubation overnight at 4°C with 2.5 µg rabbit polyclonal cdk4 antibody (Santa Cruz Biotechnology), followed by incubation for 4 hours with 25 µL protein A-agarose beads (Amersham). Precipitated protein pellets were washed 3 times with ice-cold kinase buffer (without Triton-X 100) and then resuspended in 20 µL ice-cold kinase buffer, 50 mM HEPES (pH 7.5), 80 mM -glycerophosphate, 2.5 mM ethyleneglycol bis (b-aminoethyl
ether)-N, N, N', N'-tetraacetic acid (EGTA), 10 mM
MgCl2, 1 mM dithiothreitol, 2.5 mM phenylmethylsulfonyl fluoride, 10 µg/mL aprotinin, 10 µg/mL leupeptin, and 10 mM cyclic adenosine monophosphate-dependent protein kinase-inhibitory peptide (Sigma). A total of 12 µL reaction mix containing 10 µCi (0.37 MBq) ( -32P)adenosine triphosphate (ATP;
approximately 3000 Ci/mmol [approximately 1.11 × 1014
Bq/mmol]; Amersham), 25 µM unlabeled ATP, and 200 ng
glutathione-S-transferase retinoblastoma (GST-Rb) protein as substrate (Santa Cruz Biotechnology) were added to each sample and
incubated at 30°C for 15 minutes. Kinase reactions were stopped by
the addition of an equal volume of 2× SDS sample buffer (4% SDS, 150 mM Tris-HCl [pH 6.8], 20% glycerol, 0.02% bromophenol blue, and 2 mM sodium vanadate) and by boiling for 5 minutes. Proteins were
separated by electrophoresis in 10% SDS-PAGE, gels were dried and then
autoradiographed. Radioactivity was quantified by using a Molecular
Dynamics Storm 860 phosphorimager.
PI3 kinase assays Cells were lysed in RIPA buffer at 4°C for 30 minutes. The debris was separated by centrifugation at 12 000g for 20 minutes at 4°C. Protein concentration was estimated in the cleared supernatant, and 1000 µg protein (total volume in 500 µL IP buffer) was used for immunoprecipitation. Immunoprecipitations were incubated overnight at 4°C with 2.5 µg PI3 kinase (p85) antibodies (Upstate Biotechnology, Lake Placid, NY), followed by incubation for 4 hours with 25 µL protein A-agarose beads. Precipitated protein pellets were washed 3 times with ice-cold kinase lysis buffer and 3 times with PI3 kinase buffer and then resuspended in 20 mL ice-cold kinase buffer (40 mM HEPES [pH 7.5], 2 mM EGTA, 6 mM MgCl2, 1 mM dithiothreitol, 2.5 mM phenylmethylsulfonyl fluoride, 5 mM NaCl, 0.2 mM EDTA, and 10 µM unlabeled ATP). Lipid mix (20 µL) was added (10 µg PI [Matreya, State College, PA] containing 0.5% wt/vol cholic acid, freshly prepared by sonication for 5 minutes on ice), and samples were vortexed and incubated at 30°C for 5 minutes. Then, an additional 40 µL reaction mix was added containing 10 µCi (0.37 MBq) (-32P)ATP (approximately 3000 Ci/mmol
[approximately 1.11 × 1014 Bq/mmol]; Amersham) and
incubated at 30°C for another 15 minutes. The reaction was stopped by
addition of a mixture of chloroform-methanol (1:1) and 1.5 N HCl (40 µL). The reaction was then extracted with 160 µL mixture of
chloroform-methanol at a ratio of 60:100, and appropriate washes were
performed. The extracted reaction product was combined and dried in a
vacuum centrifuge, and the residue was dissolved in 35 µL
CHCl3/MeOH 2:1 vol/vol, separated by thin-layer chromatography, and developed with
CHCl3/MeOH/NH4OH/H2O
(129:114:15:21). The dried thin-layer chromatography plates were
exposed on a phosphorimager screen, and the amount of PI3-phosphate
produced was quantified by using a Molecular Dynamics Storm 860 phosphorimager.
TEL/PDGF R transforms
IL-3-dependent Ba/F3 cells to IL-3 independence.11 To
develop a confirmatory system, we have used 32D cells, another
IL-3-dependent murine myeloid cell line.28 The 32D cells
were infected by using the pMSCV retroviral construct encoding
TEL/PDGFR complementary DNA or a control vector expressing neomycin
resistance.33 Cells were selected for growth in the
absence of IL-3. Cells infected with the TEL/PDGFR-expressing
construct grew in the absence of IL-3, but vector control cells did not
(data not shown). Expression of TEL/PDGFR was confirmed by Western
blotting (data not shown). All experiments below were repeated in both
Ba/F3-TEL/PDGFR and 32D-TEL/PDGFR cells.
TEL/PDGF R has been previously reported to associate with the
p85 subunit of PI3 kinase.11 To show that TEL/PDGFR
activates the kinase activity of PI3 kinase, PI kinase assays were
performed in transformed and untransformed Ba/F3 cells. As previously
described, short-term, high-dose stimulation of parental Ba/F3 cells
with IL-3 (50 ng/mL for 10 to 15 minutes) activates PI3 kinase (Figure 1, lane 3). The activity is blocked by
the PI3 kinase inhibitor, LY294002, as expected (Figure 1, lane 5).
Activation of PI3 kinase in cells constitutively growing in 0.5 ng/mL
IL-3 is present but low (Figure 1, lane 1). In contrast,
Ba/F3-TEL/PDGFR cells growing in log phase show high levels of PI3
kinase activity (Figure 1, lane 6). This activity is also blocked by
LY294002 (Figure 1, lane 8). To demonstrate that activation of PI3
kinase depends on the kinase activity of TEL/PDGFR, transformed
cells were incubated with the inhibitor STI571 (Figure 1, lane 10).
STI571 (previously CGP57 148B) is a well-described tyrphostin small
molecule that inhibits the kinase activity of the ABL and PDGFR
tyrosine kinases at micromolar concentrations.29,30
Inhibition of other kinases is seen with the drug but only at does 100 to 1000 times higher. The drug completely inhibits the kinase activity
of TEL/PDGFR and causes Ba/F3-TEL/PDGFR cells to undergo
apoptosis over an 18- to 30-hour period.17 As seen in
Figure 1, lane 10, STI571 inhibits the activation of PI3 kinase in
TEL/PDGFR-transformed cells growing without IL-3. To confirm the
specificity of the drug, cells were treated with STI571 and IL-3
(Figure 1, lane 11). As expected, STI571 does not inhibit the
IL-3-induced activation of PI3 kinase. Together, these data
demonstrate that TEL/PDGFR-transformed cells contain activated PI3
kinase and that this activity depends on the kinase activity of the
fusion protein.
TEL/PDGF R-transformed cells is functionally relevant, we have
examined activation of 2 mediators of the effects of PI3 kinase,
Akt/PKB (subsequently referred to as Akt), and p70S6 kinase. Akt is a serine threonine kinase that is regulated by PI3 kinase and has been
reported to lead to phosphorylation of the Bcl2 family member BAD34-36 and other proteins. As shown in Figure
2A, lane 4, TEL/PDGFR-transformed cells contain phosphorylated Akt. This phosphorylation is increased relative to that seen in parental Ba/F3 cells either growing in IL-3
(compare lane 4 with lane 1) or deprived of IL-3 (Figure 2A, lane 2).
Furthermore, the phosphorylation is inhibited by both LY294002 and
STI571 (Figure 2A, lanes 6 and 7) but not by the vehicle, DMSO (Figure
2A, lane 5). These results are consistent with a signaling pathway that
connects TEL/PDGFR with PI3 kinase and subsequently to Akt. Similar
results were seen in 32D-TEL/PDGFR cells (data not shown).
We have also examined phosphorylation of the PI3 kinase mediator, p70S6
kinase. p70S6 kinase regulates a variety of functions including
ribosomal activity. In IL-3-stimulated Ba/F3 cells, p70S6 kinase has
been reported to be necessary for the full mitogenic effect of
IL-3.37 Phosphorylation of p70S6 kinase in
TEL/PDGF Inhibition of PI3 kinase in transformed cells decreases cell growth It has been previously reported that PI3 kinase is necessary for growth of Ba/F3 cells stimulated with IL-3.24 To determine if this is true for TEL/PDGFR-transformed cells, cells were treated with LY294002, and growth curves were performed by counting the cells
daily by using trypan blue exclusion. As shown in Figure 3A, Ba/F3-TEL/PDGFR cells treated with
LY294002 at 10 µM have decreased cell growth (compare open boxes with
closed triangles). At the completely inhibitory concentration of 25 µM, cells almost completely stop growing, although they do not
undergo apoptosis for several days. In fact, examination of cultures 24 to 48 hours after addition of LY294002 shows that there is little cell
death present in treated cultures on the basis of cell morphology and trypan blue exclusion (data not shown). Sensitivity of the transformed cells to LY294002 is similar to parental cells growing in IL-3 (Figure
3B). Inhibition of cell growth in transformed cells is probably not due
to decreased activity of p70S6 kinase, as treatment of cells with
rapamycin led to only a slight and delayed decrease in cell growth in
our hands (data not shown).
Treatment with LY294002 arrests cells in the G1 phase of the cell cycle The presence of decreased cell growth with no apoptosis suggested to us that Ba/F3-TEL/PDGFR cells treated with LY294002 may be
undergoing a cell cycle arrest. To test this hypothesis, cells were
treated with LY294002 and examined for DNA content by using propidium
iodide staining at various time points. Initial analysis demonstrated
that cells began to arrest in the G1 phase of the cell cycle within 8 to 16 hours of addition of LY294002. As shown in Figure
4A, cells treated with LY294002 at 12.5 µM for 24 hours showed an accumulation of cells in the G1 phase of the cell cycle (66% versus approximately 40% or less for control cells). When LY294002 was used at 25 µM, 80% of cells arrested in
G1. This finding was true whether IL-3 was present or not, confirming
that TEL/PDGFR and IL-3 may both regulate cell cycle through a PI3
kinase-dependent pathway. Interestingly, when cells are treated with
STI571, which inactivates the kinase activity of TEL/PDGFR, a cell
cycle arrest was also seen. However, 50% of these cells have undergone
apoptosis after 24 hours (sub 2N DNA content by propidium iodide
staining), in contrast to the LY294002-treated cells (data not shown).
This finding demonstrates that there are alternative pathways to cell
survival that are activated by TEL/PDGFR and not blocked by
LY294002. Consistent with previous results of TEL/PDGFR-transformed
cells treated with IL-3 and STI571,17 IL-3 completely
rescues cells from the effects of STI571. Unlike cells transformed with
BCR/ABL, we have never seen an inability of IL-3 to rescue
TEL/PDGFR-transformed cells treated with STI571 in short- or
long-term assays. The time course of cell cycle arrest is shown in
Figure 4B. Again cells were treated with LY294002 at 25 µM. Cells
were harvested at the indicated time and analyzed for DNA content
again. Accumulation of cells in the G1 phase of the cell cycle is seen
as early as 8 hours after addition of LY294002, and cells continue to
accumulate for 24 hours.
PI3 kinase regulates cdk4 kinase in transformed cells To understand the mechanism of G1 arrest in TEL/PDGFR-transformed cells treated with LY294002, we have analyzed
activation of the cdk4 complex. In these murine hematopoietic cells,
cdk4 is a major regulator of the G1 to S phase
transition.38 Furthermore, cdk4 appears to couple with
cyclin D2 or D3 in these cells. Cyclin D1 is not expressed in these
cells at an appreciable level. To analyze cdk4 kinase activity, cells
were treated with DMSO or inhibitor for the indicated times, cells were
lysed, and immunoprecipitations were performed with cdk4 antibodies.
Immunoprecipitated complexes were incubated with purified GST-Rb fusion
protein. Rb is a known substrate of cdk4 kinase.39
Phosphorylated proteins were separated by SDS-PAGE and analyzed for
phosphorylation of GST-Rb by using a phosphoimager. Visualized
autoradiogram is shown at the top of the figure, and quantitation of
bands is graphed below. Both Ba/F3 and Ba/F3-TEL/PDGFR cells treated
with LY294002 for 24 hours show a 80% decrease in activation of the
cdk4 complex (Figure 5, lanes 2 versus 3 and 5 versus 6). No difference is seen between untreated cells and
DMSO-treated cells at this time point. Similar results are seen in 32D
and 32D-TEL/PDGFR cells (Figure 5, lanes 8-14). These results
demonstrate that inhibition of PI3 kinase in these cells leads to an
inhibition of cdk4 kinase activity.
To see if the inhibition of cdk4 kinase activity is temporally
associated with the block in the G1 phase of the cell cycle, a kinetic
analysis of cdk4 kinase activity was performed (Figure 6). Ba/F3 and Ba/F3-TEL/PDGF
Inhibition of cdk4 kinase activity is associated with decreased Cyclin D2 and increased p27Kip1 Previous reports have demonstrated a signaling cascade though which Akt activates the forkhead transcription factor, FKHR-L1, that in turn inhibits the transcription of the p27Kip1 protein and regulates progression through the G1/S cell cycle checkpoint.40 An analogous signaling cascade has also been proposed downstream of BCR/ABL in transformed Ba/F3 cells.41 To determine if the same or distinct mechanisms were regulated by TEL/PDGFR, we examined the expression of multiple
cell cycle regulatory proteins in both wild-type 32D cells and in
32D-TEL/PDGFR cells treated with either DMSO alone or LY294002. As
shown in Figure 7, Western blotting does
demonstrate an increase in p27Kip1 in IL-3-stimulated
cells (Figure 7, compare lane 1 with lanes 6 and 7). However, in
several experiments, this induction was also seen with DMSO alone
(Figure 7, lane 1 versus 3 and 4). DMSO was present at a final
concentration of 0.1%. Furthermore, in 32D-TEL/PDGFR-transformed
cells, the increase was less marked and not sustained (Figure 7,
compare lane 8 versus lanes 13 and 14). However, cyclin D2 was
consistently down-regulated by LY294002 and not by DMSO in both
IL-3-stimulated parental cells and 32D-TEL/PDGFR cells (Figure 7,
lane 1 versus 7 and lane 8 versus 14). There was also consistent
down-regulation of cyclin E (Figure 7, lanes 7 and 14). Although some
decrease in cyclin D3 is present in this experiment, this was not
reproducible (Figure 7, lane 7). Cdk4 protein level and p21 levels were
not altered in our experiments by LY294002 (Figure 7). Basal levels of
both cyclin D3 and p21 were increased in 32D-TEL/PDGFR compared with
nontransformed parental cells (Figure 7, compare lane 8 with lane 1),
but this increase was not seen in Ba/F3-TEL/PDGFR cells and is of
unclear significance. Although these data do not show that cyclin D2 is the critical regulator of cdk4 kinase activity in these cells, they do
raise the question of whether p27Kip1 is the sole cell
cycle protein regulated by the PI3 kinase/Akt pathway.
We have demonstrated that TEL/PDGF Several comments are warranted about the methods we have used. First,
we have used STI571 as a specific inhibitor of TEL/PDGF We have shown that IL-3-stimulated Ba/F3 and 32D cells arrest in G1
and down-regulate cdk4 kinase activity after treatment with LY294002
(Figures 3 and 5 and data not shown). This result is consistent with
previous results that expression of a dominant-negative p85 PI3 kinase
inhibits the growth of Ba/F3 cells,24 although this study
did not demonstrate an effect of the dominant-negative construct on
cdk4 kinase activity. The similarity in the results between
IL-3-stimulated cells and TEL/PDGF The exact signaling pathway between PI3 kinase and cdk4 kinase activity
in these cells is unclear. It has previously been reported in
fibroblasts that there is a signaling pathway from PI3 kinase through
glycogen synthase kinase 3 (GSK3) to Cyclin D1.23 GSK3
activation leads to phosphorylation of Cyclin D1 and promotion of the
G1 to S phase transition.23,42 However, Cyclin D1 is not
detectable in Ba/F3 cells. Furthermore, an experiment using LY294002
and lithium (an inhibitor of GSK3 activity) showed no change in the
cell cycle arrest seen with LY294002 alone, suggesting that this effect
may not be mediated through GSK3 (data not shown). Alternatively, it has recently been reported that IL-3 regulates the
forkhead transcription factor through PI3 kinase and that this pathway
regulates expression of the cyclin inhibitor,
p27Kip1.40 In addition, Gesbert et
al41 recently reported that BCR/ABL regulates
p27Kip1 through a PI3 kinase/Akt-dependent
pathway.41 Although we see reproducible up-regulation of
p27Kip1 in our experiments, we also see consistent down-regulation of
Cyclin D2. Additional experiments will be necessary in
TEL/PDGF Tyrosine kinase fusion proteins such as BCR/ABL, TEL/PDGF
We thank Charles Abrams and Terri Laufer for helpful discussions and Jidong Zhang for technical assistance.
Submitted May 31, 2001; accepted October 25, 2001.
Supported by grants 73747 (M.C.) from the National Cancer Institute, National Institutes of Health. M.C. is a recipient of a G&P Charitable Foundation Award.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Martin Carroll, Division of Hematology/Oncology, BRB2/3, Rm 708, 421 Curie Blvd, Philadelphia, PA 19104; e-mail: carroll2{at}mail.med.upenn.edu.
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activates
phosphatidylinositol 3 (PI3) kinase and requires PI3 kinase to regulate
the cell cycle
Top
Abstract
Introduction
B. Reports have demonstrated a direct role of the PI3 kinase/Akt
pathway in regulation of the cell cycle in both fibroblasts and
hematopoietic cells.
