|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
NEOPLASIA
From the Walther Cancer Institute and Division of
Hematology/Oncology, Departments of Medicine and Surgery, Indiana
University School of Medicine and VA Medical Center, Indianapolis, IN.
Tyrosine kinase oncogenes such as p210BCR-ABL
activate multiple signal pathways. As a result, it is difficult to
infer the functional relevance of a pathway acting alone or in
cooperation with another. One or 2 second-tier kinases represented in
the p21ras and phosphatidylinositol-3-kinase (PI-3-kinase) pathways (activated RafCAAX and gag-akt, respectively) were expressed in parental H7 interleukin-3 (IL-3)-dependent myeloid cells.
IL-3-dependent cells served, independently, as recipients of
p210BCR-ABL, which activated p21ras and PI-3-kinase pathways, including
raf/erk and akt, respectively, en route to transformation. By contrast,
neither RafCAAX nor gag-akt when expressed in parental cells in
isolation produced factor-independent cells. On the other hand, H7
cells expressing both RafCAAX and gag-akt (H7gag-akt/RafCAAX) were
transformed. Such transformation in H7gag-akt/RafCAAX was accomplished
in the absence of active versions of Shc or cbl, and there was no
evidence of Stat activity and only modest amounts of bcl-xL, a Stat5
transcriptional target protein, all of which characterized the cells
transformed by BCR-ABL. However, H7gag-akt/RafCAAX cells and
H7BCR-ABL cells cultured in the absence of IL-3 shared strikingly
increased p65 nuclear factor Considerable efforts have been devoted to
unraveling the mechanism of leukemic transformation by the p210 (or
p185) BCR-ABL product of the Philadelphia chromosome translocation in
chronic myelogenous leukemia, as well as the more commonly occurring
tyrosine kinase oncogene of acute myeloid leukemia, rendered by
internal tandem duplication (ITD) mutation of the Flt3 receptor. In the case of p210BCR-ABL, at least 3 major independent signaling pathways are simultaneously active, and these bear upon additional subsidiary pathways as well.1-19 Activity of the independent pathways
initiated by p21ras, phosphatidylinositol-3-kinase (PI-3-kinase [via
tyrosine kinase-dependent p85 adaptor
interactions]20-22), and one exerted on behalf of Stat5
has been observed in cells with the BCR-ABL protein.
Precise analysis of the individual contribution(s) of these independent
signal pathways, such as elicited by p210BCR-ABL, toward the
transformed phenotype of hematopoietic cells is quite complicated. In
part, this arises because of tyrosine kinase-dependent downstream
signal pathway cross-talk mediated via adaptor proteins. We
wished to focus on the potential for collaboration of signals emanating
from p21ras and from PI-3-kinase in hematopoietic transformation. To
further restrict the potential for cross-talk in analyzing the
collaboration of main components of these pathways (p21ras [raf/erk]
and PI-3-kinase [akt]), we used membrane-targeted active versions of
raf (RafCAAX) and akt (gag-akt) to reconstruct pathways mediated by
p210BCR-ABL in the absence of confounding adaptor-mediated cross-talk.
By the combined expression of both of these active pathways in
interleukin-3 (IL-3)-dependent hematopoietic cells, but not when
either was present alone, hematopoietic factor-dependent cell lines
were transformed to growth factor independence and achieved continuous
but slow proliferation, and apoptosis was retarded. These effects were
linked to the activity of p65 NF Genes and vectors
Cells and cell culture
Where appropriate, cells were treated with various concentrations of
parthenolide, a sesquiterpene lactone-specific inhibitor of NF Cell lysates Preparation of cell lysates for assay of cytosolic or nuclear proteins was performed as described previously.29,30Immunoblotting Cytosolic or nuclear proteins were resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose filter by a semidry Western blotting method (BioRad, Hercules, CA). Immunoblotting was performed as described previously.29,30Erk kinase assay Cellular proteins, 250 µg/reaction, were diluted to 1 µg/mL in phosphotyrosine lysis buffer and subjected to immunoprecipitation with 5 µg antimouse ERK antibody (Santa Cruz Biotechnology, Santa Cruz, CA) along with protein G PLUS agarose (Oncogene Science, Uniondale, NY) by gentle rocking at 4°C. The agarose beads were washed twice with lysis buffer and then once with kinase buffer (30 mM Tris HCl, pH 8, 20 mM MnCl2, 2 mM MgCl2, 10 µM ATP). The pellet was resuspended in 30 µL kinase buffer along with 7.5 µg myelin basic protein (MBP; Sigma, St Louis, MO) and 37 kBq (1 µCi) 32P-ATP.30 The
kinase reaction was allowed to proceed at 37°C for 30 minutes, after
which the reaction was stopped by addition of Laemmli sample buffer.
The reaction contents were boiled and subjected to electrophoresis on
15% SDS-polyacrylamide gels. Phosphorylated proteins were visualized
by autoradiography of the dried slab gels.
Akt kinase assay Cellular proteins, 200 µg, were immunoprecipitated with 2.5 µg anti-protein kinase B (PKB) (Upstate Biotechnology, Lake Placid, NY) and 15 µL protein G PLUS agarose (Oncogene Science) after gentle rocking at 4°C for 3 hours. After pelleting at 2500 rpm for 5 minutes, the beads were washed twice with lysis buffer, once with H2O, and once with PKB kinase buffer, while maintaining the beads on ice at all times. Finally, the beads were resuspended in 25 µL PKB kinase buffer (20 mM HEPES, 10 mM MgCl2, 10 mM MnCl2) and exposed to substrate, histone H2B (Roche Biologicals, Indianapolis, IN), 2.5 µg/reaction, in a final volume of 30 µL along with 32P-ATP, 92.5 kBq (2.5 µCi), in a final concentration of cold/radioactive ATP, 1.25 µM/2.5 µM. The reaction was allowed to proceed at room temperature for 30 minutes with occasional agitation. The reaction was then stopped with SDS-PAGE sample buffer, boiled, subjected to electrophoresis on 15% polyacrylamide, and autoradiographed.Gel mobility shift analysis Cell nuclear protein lysates, 5 to 15 µg, prepared by the above protocol were placed in the electrophoretic mobility shift assay with oligonucleotide probe for NF B site (Promega Biotech, Madison,
WI), using methods described previously.30
Antibodies The following antibodies were used for supershifting (gel mobility shift assays) or immunoblotting of proteins Western transferred onto nitrocellulose filters after SDS-PAGE: anti-Akt-1 (clone D-17), anti-Erk1 (691) (sc-94), anti-Raf C-terminal portion (clone C-12), anti-Raf N-terminal portion (sc-134) and Raf internal part (clone H-71), anti-c-myc (clone 9E10), anti-cbl (sc-170), anti-Stat5 (sc-1081), anti-NFB p50 (sc-7178),
anti-c-Rel (sc-272), and anti-Rel-B (sc-226) (Santa Cruz
Biotechnology). Anti-phospho-Tyr (clone 4G10), anti-Shc (#06-203),
anti-Akt/PKB (#06-558), and anti-NFB p65 (#06-409) were from
Upstate Biotechnology. Antirabbit glyceraldehyde-3-phosphate dehydrogenase (GAPDH; H86504) was from Biodesign International (Kennebunk, ME). Rabbit anti-polyADP ribosyltransferase
(PARP) antibody was from Enzyme Systems Products (Livermore,
CA). Horseradish peroxidase-conjugated antimouse IgG
(sc-2005), antirabbit IgG (sc-2005), and antigoat IgG
(sc-2020) were from Santa Cruz Biotechnology. Horseradish
peroxidase-conjugated rabbit anti-sheep IgG (#6134328) was
from Rockland (Gilbertsville, PA). Fluorescein isothiocyanate-labeled annexin V protein was from Becton Dickinson/Pharmingen
(Thousand Oaks, CA).
Expression of gag-akt and/or RafCAAX within IL-3-dependent cell line H7 leads to selective augmentation of enzymatic activity in that pathway targeted by the transfected enzyme Stable transfections into parental H7 cells were accomplished by electroporation followed by selection of cells cultured in liquid phase, in the presence of the relevant antibiotic for which resistance was encoded by the vector used. First, H7 cells were transfected with pBabgag-akt, whose stable genomic incorporation and expression were enforced by selection of transfectants in the presence of puromycin, 5 µg/mL. Expression was verified by immunoblotting with an akt antibody (see below). Next, either H7 cells or H7gag-akt cells were transfected with pRafCAAX/LXSN, and transfectants were selected in the presence of G418, 500 µg/mL, or in the presence of puromycin and G418, respectively. H7 cells were also transfected with pGD210BCR-ABL.30Lysates of the respective cell lines were first immunoblotted for the
detection of akt and gag-akt (Figure 1).
The gag-akt fusion species of 96 kd was observed only in
lysates of those cells transfected with the vector pBabgag-akt, and it
was observed that the amount of this species immunoreactive with the
anti-akt antibody appeared to represent a significantly smaller
fraction than the endogenous akt species of 66 kd (Figure 1). In fact, the amount of the 96-kd gag-akt species was variably represented from
different lysates of the same cell line, indicating its highly unstable
nature in lysates (Figure 1). Notably, however, it was repeatedly
observed in these cell lines transfected with pBabgag-akt and
expressing variable amounts of the 96-kd fusion protein that the amount
of the presumed endogenous 66-kd akt species was significantly increased 2- to 3-fold as compared with equal amounts of lysates from
the nontransfected cells (Figure 1). This was confirmed by reprobing
the blots with antibody for a constitutively expressed protein as
loading control, GAPDH (Figure 1). Thus, we were led to conclude that
gag-akt is initially placed in the membrane of cells containing the
construct encoding its expression, where it is capable of
phosphorylation by the PDK activating kinases, and it is subsequently
also observed in cell lysates as an immunoreactive cleaved species with
the same apparent molecular weight as endogenous akt. This
interpretation was further supported by immunoprecipitation experiments
(data not shown).
We next immunoblotted lysates for detection of RafCAAX, a task that was facilitated by the presence of an N-terminal myc tag encoded in the vector (Figure 1). Cell lysates were immunoblotted with 9E10 monoclonal antibody, raised specifically against the myc tag epitope. Both H7RafCAAX and H7gag-akt/RafCAAX expressed the 72-kd RafCAAX with the myc tag (Figure 1). Interestingly, when 9E10 immunoprecipitates were probed with an antibody against the N-terminal domain of c-raf, but not with a C-terminal antibody, selective detection of the 72-kd Raf epitope was observed in these same lysates (data not shown). This is because the C-terminal epitope of endogenous c-raf is blocked by the CAAX box in pRafCAAX/LXSN, resulting in failure of detection of the C-terminal epitope (Figure 1). Thus, taken in total, we had cell lines expressing either RafCAAX or gag-akt, or both these species, and the relative equality of expression of these respective proteins, when comparing the cell lines transfected with both vectors versus those transfected with only a single species, was fortified by reprobing blots for 2 different protein loading controls, using GAPDH and C-terminal (endogenous) c-raf antibodies (Figure 1). Next, kinase assays were performed to examine activity for signaling
within the pathways targeted by the exogenous proteins expressed by the
vectors. First, we performed akt immune complex kinase assays to
distinguish the akt activity between cells transfected with the gag-akt
vector versus those transfected with the RafCAAX vector, where, by
comparison, one would expect to observe augmented akt activity in the
former. In this experiment, NIH 3T3 cells passaged in regular medium
versus the same cells acutely stimulated with epidermal growth factor
(EGF), a strong akt agonist, served as controls (Figure
2).
Unstimulated NIH 3T3 cells had no discernable akt activity, whereas EGF stimulated abundant activity of akt for the substrate H2B within NIH 3T3 cells (Figure 2). Similarly, H7RafCAAX cells in regular passage medium in nonexponential growth had little measurable akt kinase activity, but the H7gag-akt/RafCAAX cells had strong activity of the kinase for H2B substrate (Figure 2). A control lane in which H7gag-akt/RafCAAX lysate was treated with nonimmune immunoglobulin lacked the activity seen in the specific immunoprecipitate (Figure 2). In other experiments, we studied the kinase activity of akt in IL-3-dependent cells acutely stimulated with WEHI-3 CM, and we observed that 3- to 5-fold increases in akt activity could be stimulated by treatment (data not shown). In addition, we compared the akt activity ratios between BCR-ABL.A54 cells versus H7 parental cells starved of IL-3, versus H7gag-akt cells similarly starved, and we observed comparable activity of akt within cells with p210BCR-ABL and those with membrane-targeted, constitutively active akt, both of which were 2- to 3-fold greater, respectively, than a background level of activity in the starved H7 parental cells (data not shown). We next conducted a complementary analysis of activity in the raf/erk
pathway using a well-characterized erk kinase assay with MBP substrate
(Figure 3). First, a series of the cells
with or without RafCAAX or gag-akt were depleted of growth factors, and
lysates were prepared and immunoprecipitated with anti-erk, followed by
washing and immune complex kinase assay using MBP substrate (Figure 3).
BCR-ABL.A54 cells had abundant erk activity, whereas H7 parental cells
starved of IL-3 had little activity and H7gag-akt cells similarly
starved also lacked kinase activity of erk (Figure 3). By contrast,
there was greater erk activity apparent in the H7RafCAAX cells, and
even more kinase activity of erk, comparable to that observed in
BCR-ABL.A54 cells, was observed in H7gag-akt/RafCAAX lysates (Figure
3). We also observed strong stimulation of erk kinase activity by IL-3
stimulation of the parental cells and H7gag-akt cells, and the higher
basal levels of erk activity seen in the presence of IL-3 in medium supporting factor-dependent cells were verified by blotting lysates with anti-phospo-erk antibodies (Figure 3 and data not shown).
No evidence was found for adaptor protein (shc, cbl) or Stat5 involvement in cells transformed by combined expression of gag-akt and Raf-CAAX We conducted a formal analysis within the H7gag-akt/RafCAAX cells for the possible existence of activated forms of adaptors implicated in p21ras and PI-3-kinase/akt activation initiated by tyrosine kinase oncogenes or growth factor receptors. For this purpose, we used BCR-ABL.A54 cells and IL-3-starved H7 parental cells as positive and negative controls, respectively. This was done to support selectivity of downstream signaling by the transfected membrane-active kinases and to better prove functional independence of the cooperative activity of gag-akt and RafCAAX toward perpetuation of a factor-independent state of growth.First, whole-cell lysates of starved cells were immunoblotted
with antiphosphotyrosine to examine for possible constitutive tyrosine
signaling within H7gag-akt/RafCAAX cells that might belie autonomous
function of the membrane-active downstream kinases (Figure
4A, right panel). As compared with
BCR-ABL.A54, which displayed prominent phosphotyrosine bands at 210, 110, 52, and 35 kd, parental H7 cells and H7gag-akt/RafCAAX cell
lysates displayed only a prominent 145-kd band, putative SHIP (Figure
4A, right panel). Next, cell lysates were immunoprecipitated with
anti-Shc and then immunoblotted with antiphosphotyrosine to look for an "active bait" Shc species that could participate in recruitment of
Grb-2 or PI-3-kinase (Figure 4A). In BCR-ABL.A54 cell lysates, there
was abundant tyrosine-phosphorylated Shc that was immunoprecipitated, whereas in the H7gag-akt/RafCAAX and in the H7 parental cell lysates, no tyrosine-phosphorylated Shc was observed despite quantitatively equal immunoprecipitation of total Shc protein in each lysate, as
revealed by reprobing the blot with anti-Shc (Figure 4A, bottom). In
addition, anti-Shc immunoprecipitates from the BCR-ABL.A54 cell lysates
coprecipitated a tyrosine-phosphorylated 145-kd species, putative SHIP
(Figure 4A). Similarly coprecipitated with Shc from the H7 parental and
H7gag-akt/RafCAAX lysates was the same tyrosine-phosphorylated putative
SHIP species, which is known to be bound, when it is tyrosine
phosphorylated, by Shc PTB domain (Figure 4A).
Next, lysates of the above cell lines were immunoprecipitated with anti-cbl that targets an adaptor, when tyrosine phosphorylated, implicated in PI-3-kinase activation by binding to p85 SH2s (Figure 4B). In fact, anti-cbl immunoprecipitates of BCR-ABL.A54 cells contained abundant tyrosine-phosphorylated cbl and a coprecipitating Ptyr 49-kd species, putative Shc (Figure 4B). By contrast, there was little tyrosine-phosphorylated cbl detected in the anti-cbl immunoprecipitates of lysates from H7 parental cells or H7gag-akt/RafCAAX, despite comparable efficiency of immunoprecipitation (Figure 4B). We also confirmed the absence of tyrosine-phosphorylated, active Stat5 in lysates of H7gag-akt/RafCAAX cells grown in the absence of IL-3 (Figure 4C). Unlike BCR-ABL.A54 cell lysates, which contained abundant tyrosine-phosphorylated Stat5a detectable in the absence of IL-3, H7gag-akt/RafCAAX contained active Stat5 only upon IL-3 stimulation (Figure 4C). In this experiment, IL-3-starved H7 parental cells similarly lacked active Stat5, and stimulation of phospho-Stat5 by IL-3 was not seen at the relatively late time point used (60 minutes); however, another experiment demonstrated stimulation of Stat5 phosphorylation at 5 to 30 minutes (data not shown). Thus, taken in total, cells expressing both gag-akt and RafCAAX were capable of growth in the absence of IL-3 (see below), and this state of competence for growth was accomplished in the absence of any discernable stimulus, in the form of phosphotyrosine-initiated, adaptor-mediated parallel signaling, from above the level of these second-tier kinases. We next established the parameters for growth of cells containing one or both of these kinases as compared with the fully transformed BCR-ABL.A54 cells. Competence for factor-independent growth of H7gag-akt/RafCAAX cells
differs from that of cells with p210BCR-ABL in degree, but common
functional end points are summoned, including NF
Cells transformed by p210 BCR-ABL doubled frequently and achieved very high culture density of more than 1.5 million cells/mL (Figure 5). By contrast, after IL-3 deprivation in either low serum or in 10% serum, all of the following cell lines rapidly died off: H7 parental, H7gagakt, and H7 (ectopic) cyclin D1-expressing (control) cells. On the other hand, H7RafCAAX cells persisted for 4 to 5 days in 1% FCS before dying off entirely, but they persisted somewhat longer in 10% FCS. However, H7gagakt/RafCAAX cells, removed from IL-3 and serum, maintained their viable cell numbers indefinitely, and in 10% FCS they grew continuously (Figure 5). Inhibition of apoptosis in H7 cells with RafCAAX or gag-akt/RafCAAX was confirmed by analysis of the subG0 DNA content of propidium iodide-loaded cells by fluorescence-activated cell sorter analysis (data not shown). As might be predicted from these results of proliferation, expression of the nuclear c-myc gene, whose transcripts were detected on Northern blot (and normalized for a constitutively expressed control gene, GAPDH), was not so strong in H7gagakt/RafCAAX cells as in p210BCR-ABL-transformed cells (data not shown). Nor was expression of cyclin D1 protein as great in cells with H7gag-akt/RafCAAX as in p210BCR-ABL (data not shown). Thus, cell cycle-dependent proteins were understimulated in H7 cells transformed with the combination of gag-akt/RafCAAX compared with BCR-ABL. We also observed the ability of the combination of gag-akt/RafCAAX to result in factor-independent slow proliferation of the FDC-P1 cell line (V. Gelfanov, H. S. Boswell, unpublished observations, 2000). The uniform ability of this oncogene (akt/raf) combination to mediate a hypoproliferative transformed state in different cell lines suggested that some other function rather than excessive cell-cycle transit alone would be foremost in the transforming program. A recently appreciated target for downstream signal integration, known
to contribute to both inhibition of apoptosis and proliferation induction, is NF First, gel mobility shift analysis was performed to search for
the previously observed p65 heterodimeric species of NF
Nuclear protein extract from H7gag-akt/RafCAAX cells, when placed in
gel mobility shift assay alongside that described above, showed the same 2 separately migrating complexes bound to NF Assessment of the relative abundance of the 2 aforementioned
complexes in the cell lines taken after IL-3 deprivation or, alternatively, from their normal culture medium containing 10% FCS
(BCR-ABL.A54 cells and H7gag-akt/RafCAAX cells cultured without IL-3
required) showed the lowest ratio of the putative p65 heterodimeric species/p50 homodimeric species in the H7 parental cells (Figure 7A, left). The highest such ratio was
observed in H7gag-akt/RafCAAX1 and H7gag-akt/RafCAAX2 cells (Figure
7A). Next in rank order was the relative p65 NF
Inhibition of NF B from other
pathways downstream from p210BCR-ABL, it was of interest to compare the
consequences of loss of functional NFB expression in cells with
Stat5 and NFB (BCR-ABL.A54) versus cells with only NFB (H7gag-akt/RafCAAX). We used a specific inhibitor of NFB,
parthenolide, a sesquiterpene lactone,28 in preliminary
experiments to study the consequences of specific NFB inhibition and
performed control assays of dose- and time-dependent viable cell
recovery and corresponding inhibition of NFB by gel shift. We found
that there was a relation between parthenolide dose and inhibition of
viable cell recovery, with a requirement for approximately 17 to 24 hours for cell killing to occur at all doses of parthenolide up to 10 µM (IC50 approximately 5 µM; data not shown). At a
parthenolide concentration of 10 µM, loss of viable cell recovery of
H7gag-akt/RafCAAX1 was preceded by significant inhibition of specific
NFB binding at 4 hours (greater than 75% inhibition of binding to
specific NFB oligo by gel shift in both H7gag-akt/RafCAAX1 and also
BCR-ABL.A54; data not shown). However, doses of parthenolide
up to 40 µM for 24 hours did not reduce the viability of BCR-ABL.A54
beyond background, whereas H7gag-akt/RafCAAX1 cells were reduced by
85%. In addition to viability counting, 2 measures of apoptosis were
determined: annexin V staining for exteriorized phosphatidylserine and
cleavage of PARP, a caspase-3 target (Figure
8). By both of these measures, BCR-ABL.A54 cells were refractory to the parthenolide treatment, which
resulted in dramatic apoptosis in H7gag-akt/RafCAAX cells (Figure 8).
Simultaneous assay of specific caspase-mediated apoptosis (cleavage of
PARP) and measurement by immunoblotting of c-IAP2 (an NF
An extensive descriptive analysis of the mechanism for
hematopoietic transformation by representative leukemic tyrosine kinase oncogenes p210BCR-ABL, mutant (ITD) Flt3, and TelPDGF has
been presented. Simultaneous activity of multiple upstream and
downstream effectors has been described, including, respectively,
active p21ras, raf, PI-3-kinase, and akt, then followed downstream by c-myc, cyclin D1, c-jun/AP-1, Stat5, and, more recently,
NF Left to be fully addressed, then, is the question of what is the
minimal cooperation needed among other major pathways identified in the
cell transformed by tyrosine kinase oncogenes such as p210BCR-ABL to
achieve independence from hematopoietic growth factor(s) for antiapoptosis, rendered by a defined target protein. It was with this
goal in mind that we coexpressed active versions of second-tier kinases
in the p21ras and PI-3-kinase pathways within the same parental cells
that were also independently engineered to express p210BCR-ABL; in the
latter situation, we and others have noted activity of p21ras and
PI-3-kinase.1,8-11,45,46 Ultimately, we discovered that
raf and akt expressed together in cells, each in an active form, was a
relatively pure cooperative stimulus to p65 NF In fact, we observed that coexpression of gag-akt and RafCAAX rendered IL-3-independent proliferation to H7 factor-dependent parental cells, and this state was accomplished in the absence of confounding signaling from upstream tyrosine kinases or their subservient adaptor signal amplifiers. This state was also independent of the downstream transcriptional mediator Stat5, previously demonstrated to contribute functionally to p210BCR-ABL transformation.12-15,31 In addition, our study found that the end point effectors cyclin D1 and c-myc (data not shown) were not fully reconstituted to the level elicited by p210BCR-ABL even upon cooperation of gag-akt with RafCAAX. Apparently as a direct result, the proliferative rate of H7gag-akt/RafCAAX cells was significantly blunted compared with those H7 cells with p210BCR-ABL. However, our study has more important implications in the clarity it
provides for viewing the potential antiapoptotic programs downstream
from certain tyrosine kinase oncogenes such as p210BCR-ABL. In fact,
the absence of significant Stat5 and bcl-xL activity in
H7gag-akt/RafCAAX cells allows one to view a parallel antiapoptotic pathway in isolation. We demonstrated here that such a pathway at a
minimum includes the cooperation of raf/erk and akt to elicit p65
NF One may ask how cooperative interaction of raf/erk and akt
activates NF Finally, our study also distinguished the major species of NF In summary, the present report focuses on the functional effect of
cooperation between 2 pathways (raf/erk and akt) known to operate
downstream from p210BCR-ABL (as a possibly representative tyrosine
kinase oncogene). The study demonstrates that the combination of active
versions of raf and akt can transform hematopoietic cells by an
antiapoptotic and hypoproliferative effect through NF
We acknowledge the gift of reagents by Drs Albert S. Baldwin, Jr, B. M. Th. Burgering, and D. Beach, as well as assistance from Dr Nikkil Patel and helpful discussions from Elizabeth Smith.
Submitted October 30, 2000; accepted June 6, 2001.
Supported in part by a Merit Review Award from the Department of Veterans Affairs and by the Leukemia and Lymphoma Society (formerly Leukemia Society of America) (to H.S.B.) and by American Cancer Society grant RPG-00-122-01-TBE (to H.N.).
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: H. Scott Boswell, Division of Hematology/Oncology, Indiana University Medical Center, Rm 202, R4 Building, 1044 West Walnut St, Indianapolis, IN 46202; e-mail: hboswell{at}iupui.edu |
B-mediated antiapoptosis involving
c-IAP2
Top
Abstract
Introduction
