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Experimental Therapeutics |
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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7.9 kDa from the
untreated cells and two phosphorylated fragments, of 7.9 and 9.6
kDa, from the Taxol-treated cells. The domain of Taxol-induced serine
phosphorylation is thought to be in the cyanogen bromide
fragment containing residues 2–65. The Taxol-induced electrophoretic
mobility shift of p66shc was inhibited by the protein synthesis
inhibitor, cycloheximide, but not by the mitogen-activated and
extracellular signal-regulated protein kinase kinase (MEK)
inhibitor, PD98059. This mobility shift did not occur in
Taxol-resistant A549-T12 cells treated with 20 nM Taxol. In
addition to Taxol, other microtubule-interacting drugs caused a
decreased electrophoretic mobility of p66shc. This Taxol-mediated
serine phosphorylation seen in p66shc may result from a MEK-independent
signaling pathway that is activated in cells that have a prolonged or
abnormal mitotic phase of the cell cycle and may play a role in
signaling events that lead to cell death. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In addition to its effects on microtubules, Taxol, particularly in
mouse macrophages, induces the production of cytokines, such as tumor
necrosis factor 
and interleukin 1, and increases tyrosine
phosphorylation of proteins, including
MAP3
kinase (10, 11, 12, 13, 14)
. It has been reported that Taxol- and
LPS-induced activation of mouse macrophages is mediated by heat shock
protein 90 (15)
. CD18 also has been shown to be a cellular
target for Taxol in murine macrophages (16)
. In other
studies using phage-displayed peptides, Bcl-2 has been identified as a
Taxol binding protein (17)
.
Several lines of evidence indicate that Taxol can activate a variety of signal transduction pathways, e.g.: (a) Taxol activates c-Jun NH2-terminal kinase through both Ras and apoptosis signal-regulating kinase 1 pathways (18 , 19) . c-Jun NH2-terminal kinase activation mediates both Taxol-induced gene expression and cell death (20) ; (b) Taxol induces activation of Raf-1 and ERK (11 , 19 , 21, 22, 23, 24) ; and (c) Taxol causes tyrosine phosphorylation of p38 in cells of monocytic origin (25) and induces activation of p38 in human breast cancer lines (26) . In addition, tyrosine phosphorylation of Shc and formation of a Shc/Grb2 complex have been demonstrated in Taxol-treated murine macrophage RAW 264.7 cells (23) .
The SH2-containing Shc proteins (27) are substrates of activated tyrosine kinases and have been implicated in a signaling cascade leading to Ras activation (28) . Shc proteins are phosphorylated by receptor tyrosine kinases, such as EGFR (27) , insulin receptor (29) , and erbB-2 (30) , and by cytoplasmic tyrosine kinases, such as activated Src, Fps, and Lck (31 , 32) . Ligand binding induces autophosphorylation of receptors, and the activated receptors associate with and phosphorylate Shc through Shc SH2 and/or phosphotyrosine binding domains (33) . Phosphorylation of Shc proteins generates a docking site for the SH2 domain of Grb2 (34) that in turn interacts with SOS, the guanine nucleotide exchange factor for Ras, through its SH3 domains. The formation of a Shc/Grb2/SOS complex results in the membrane relocalization of SOS and activation of Ras (35) .
Shc is expressed as three proteins of 46, 52, and 66 kDa (27) . The 46- and 52-kDa isoforms result from use of distinct initiation sites in the same transcript, whereas the 66-kDa isoform is believed to arise from an alternatively spliced message (27) . All three isoforms have been structurally characterized (27 , 36 , 37) . The 52- and the 46-kDa isoforms share a COOH-terminal SH2 domain, a collagen-homologous, glycine/proline-rich region (CH1) and an NH2-terminal phosphotyrosine binding domain. The predicted amino acid sequence of the 66-kDa isoform overlaps with that of the 52-kDa isoform and contains a unique NH2-terminal region, CH2, which is also rich in glycines and prolines (36 , 37) . The 66-kDa Shc, unlike the 52- and 46-kDa Shc isoforms, is unable to transform mouse fibroblasts when expressed in vitro (36) . In addition to tyrosine phosphorylation of the Shc isoforms, it has been reported that insulin and EGF cause an increase in serine/threonine phosphorylation of the 66-kDa Shc protein (37 , 38) . Unlike the 52- and 46-kDa Shc, the 66-kDa Shc is a negative regulator of the EGF-stimulated MAP kinase-fos signaling pathway, and its CH2 domain mediates this inhibitory effect (36) . Recently, it has been reported that p66shc controls the oxidative stress response and that a targeted mutation at the serine residue in the CH2 domain of the mouse p66shc gene induces stress resistance and prolongs life span (39) .
We have demonstrated previously that Taxol induces tyrosine phosphorylation of Shc, particularly the 52-kDa isoform, and formation of a Shc/Grb2 complex in the murine macrophage cell line RAW 264.7 (23) . The aim of this study was to determine the involvement of Shc in Taxol-treated, non-macrophage-like human cell lines. We found that Taxol caused a decreased electrophoretic mobility of p66shc in the human lung carcinoma cell line A549. This shift was not the result of tyrosine phosphorylation; rather, Taxol mediated an altered serine phosphorylation of p66shc. There was no indication of a Shc/Grb2 complex in these Taxol-treated cells. These results suggest that Taxol, at nanomolar concentrations and over a 9–18-h period, induces a signaling pathway that is distinct from one that involves immediate activation of tyrosine kinases by micromolar concentrations of Taxol, such as was reported in mouse macrophages (23) .
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MATERIALS AND METHODS |
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Preparation of Cell Lysates.
Cells were washed three times with PBS, lysed with boiling buffer
containing 1% SDS, 10 mM Tris-HCl (pH 7.4), boiled for an
additional 5 min, passed six times through a 26-gauge needle, and
centrifuged for 5 min in a microcentrifuge. Such denatured lysates were
mixed with SDS-sample buffer and analyzed by SDS-PAGE. Nondenatured
lysates were prepared by incubating cells in buffer A containing 1%
Triton X-100, 150 mM NaCl, 10 mM Tris-HCl (pH
7.4), 1 mM EDTA, 1 mM EGTA, 0.2 mM
sodium vanadate, 0.2 mM phenylmethylsulfonyl fluoride, and
0.5% NP40 at 4°C for 30 min. In some experiments, 1 mM
NaF was included in buffer A. Aggregates were dispersed by passing
through a 21-gauge needle five times, and insoluble material was
removed by centrifugation for 15 min at 3000 rpm at 4°C.
Immunoprecipitation.
Equivalent amounts of protein (200–400 µg) of nondenatured lysates
were precleared by incubating with 20 µl of protein A-agarose beads
for 1 h at 4°C, followed by centrifugation at 13,000 rpm. The
supernatants were incubated with specific primary antibodies in buffer
A overnight at 4°C. Protein A-agarose beads were added and incubated
for an additional 1 h at 4°C. Immunocomplexes were collected by
centrifugation, washed five times with buffer A, eluted with boiling
SDS-sample buffer, and analyzed by SDS-PAGE.
Western Blot Analysis.
Total cell lysates or immunoprecipitates were resolved by SDS-PAGE and
transferred to nitrocellulose. Western blot analysis was performed as
described (23)
with ECL reagent. In some experiments,
blots were stripped in 100 mM ß-mercaptoethanol, 2% SDS,
and 62.5 mM Tris-HCl (pH 6.7) for 30 min at 55°C,
reblocked, and reprobed.
Two-Dimensional Gel Electrophoresis.
Approximately 40 µg of nondenatured lysate was resolved by
two-dimensional gel electrophoresis as described by O’Farrell
(41)
. Proteins were transferred to nitrocellulose, and
Western blot analysis was done with monoclonal anti-Shc antibody.
Protein Phosphatase Treatment.
Cell lysates were immunoprecipitated with polyclonal anti-Shc antibody,
and the immunoprecipitate/protein A agarose was incubated with 0.25
µg of protein phosphatase 2A1 in a buffer containing 25
mM Tris-HCl (pH 7.0), 0.2 mM
MnCl2, 1 mM DTT, and 0.1 mg/ml BSA
for 1 h at 30°C. The reaction was stopped by heating at 100°C
in sample buffer prior to analysis by SDS-PAGE.
32P-Labeling of Intact Cells and Immunoprecipitation
of Shc Proteins.
Cells were grown in complete medium for 12 h in the presence and
absence of Taxol. Medium was aspirated and replaced with phosphate-free
RPMI 1640 and fresh Taxol. After 1 h,
[32P]Pi was added (0.4
mCi/ml) and incubated for an additional 4 h. Cells were washed
twice with cold saline (0.9% NaCl) and lysed with 1% SDS, 10
mM Tris-HCl (pH 7.5). Lysates were diluted with water and
2x nondenaturing lysis buffer (final SDS concentration, <0.5%) and
immunoprecipitated with polyclonal anti-Shc antibody. The
immunoprecipitates were analyzed by SDS-PAGE.
Cyanogen Bromide Digestion of p66shc.
32P-Labeling of intact A549 cells and
immunoprecipitation of lysates with anti-Shc antibody were performed as
described above. Immunoprecipitates were resolved by SDS-PAGE and
transferred to nitrocellulose. 32P-Labeled p66shc
was visualized by autoradiography, and the appropriate area of the blot
was carefully excised. The nitrocellulose strips were incubated with
100 mg/ml CNBr in 70% formic acid for 3 h at room temperature.
The supernatant was taken to dryness in a Speed-vac, and the residue
was dissolved and re-evaporated three times with water. Phosphopeptides
were resolved on a 15% Tricine gel with 0.1 M Tris, 0.1
M Tricine, and 0.1% SDS as the cathode buffer
(42)
, transferred to nitrocellulose, and subjected to
autoradiography.
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RESULTS |
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. No mobility shift was seen for either p52shc or p46shc.
Similar results were obtained with HeLa and SKOV3 cells treated with
Taxol (data not shown).
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. It has been demonstrated that reduction in
electrophoretic mobility of Raf-1 after Taxol treatment correlated with
the activation of Raf-1 kinase activity in A549 cells
(24)
. Taxol also caused the phosphorylation of ERK1/2 in a
dose-dependent manner, as detected by an antibody specific for the
phosphorylated form of ERK1/2 (Fig. 1C)
. In addition, Taxol,
>10 nM, induced cleavage of PARP to its
NH2-terminal 24-kDa and COOH-terminal 89-kDa
fragments (Fig. 1, D and E)
. PARP is one of the
earliest proteins targeted for specific cleavage during apoptosis
(43)
. Therefore, the Taxol-induced electrophoretic
mobility shift of p66shc coincided with phosphorylation of Raf-1 and
ERK1/2, as well as with cleavage of PARP. Previous studies with A549
cells indicated that apoptosis occurred in 25% of the cells after
an 18-h exposure to 12 nM Taxol
(24)
.
Although phosphorylation of ERK1/2 coincided with the cleavage of PARP,
it was not clear whether ERK1/2 activation mediated apoptosis. The MEK
inhibitor PD98059, which blocks the activation of MEK, and UO126, which
inhibits both active and inactive MEK, clearly inhibited Taxol-induced
phosphorylation of ERK1/2 but did not prevent Taxol-mediated PARP
cleavage (Fig. 2)
. These results indicated that Taxol-induced PARP cleavage was not
mediated by activated ERK1/2. It has been reported in leukemia cells
that microtubule-active drugs induced apoptosis, which was associated
with Raf-1 and Bcl-2 phosphorylation but independent of the MAP kinase
pathway (44)
. Similar results were found in MCF-7 cells
(45)
.
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4 h after the sensitive
A549 cells were treated with 20 nM Taxol, reaching a
maximum at approximately 9–18 h and starting to decline at 21 h
after drug treatment (Fig. 3
, upper panel). An essentially identical time course was
obtained for Taxol-induced phosphorylation of Raf-1 (Fig. 3
,
lower panel).
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. These results were determined by immunoprecipitation of cell lysates
prepared from control and Taxol-treated A549 cells with polyclonal
anti-Shc antibodies, followed by Western blot analysis with monoclonal
anti-phosphotyrosine (Fig. 4A
, right panel) or
anti-Grb2 antibody (Fig. 4B
, right panel). Equal
levels of Grb2 were found in the cell lysates prepared from control and
treated cells (data not shown). The blots were stripped, reblocked, and
reprobed with anti-Shc monoclonal antibody to ensure equal loading of
immunoprecipitated protein (Fig. 4C)
. The reciprocal
immunoprecipitation using recombinant anti-phosphotyrosine antibody
RC20, followed by Western blot analysis with monoclonal anti-Shc
antibody, also demonstrated that Shc proteins were not tyrosine
phosphorylated (data not shown). As positive controls, serum-starved
A549 cells were treated with EGF for 5–15 min, and the lysates were
immunoprecipitated with anti-Shc antibody. Western blot analysis using
anti-phosphotyrosine and anti-Grb2 antibodies clearly demonstrated that
Shc proteins were tyrosine phosphorylated (Fig. 4A
,
left panel), and the Shc/Grb2 complex was formed upon EGF
treatment (Fig. 4B
, left panel). As expected, EGF
also caused tyrosine phosphorylation of the EGFR (Fig. 4D)
and EGFR/Shc complex formation (Fig. 4E
, left
panel), results not obtained after Taxol treatment (Fig. 4E
, right panel).
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). To determine the nature of the p66shc modification, the
Taxol-induced p66shc electrophoretic mobility shift was analyzed
initially by two-dimensional gel electrophoresis. Nondenatured lysates
prepared from control and Taxol-treated A549 cells were separated by
isoelectric focusing followed by SDS-PAGE (41)
. The
resolved proteins were analyzed by immunoblotting using monoclonal
anti-Shc antibody (Fig. 5A)
. No major differences were observed for p52shc and p46shc
between control and Taxol-treated cells (data not shown). p66shc was
visible as a poorly resolved charge train in control cells. Treatment
with Taxol (50 or 100 nM) resulted in an altered
charge train that was more negatively charged (Fig. 5A)
. In
addition, serine/threonine PP2A1 inhibited the electrophoretic mobility
shift of p66shc (Fig. 5B)
. It is known that PP2A is
associated with microtubules and is regulated during the cell cycle
(46
, 47)
. Protein phosphatases 1 and 2B had no effect on
the shifted form of p66shc at the concentrations tested (data not
shown).
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. p66shc was phosphorylated in both control and
Taxol-treated cells, but the Taxol treatment resulted in a decreased
electrophoretic mobility of the phosphorylated p66shc. The levels of
p66shc phosphorylation were approximately 30–70% higher in the
Taxol-treated cells than in the control cells. Both the original and
the shifted p66shc were predominantly serine phosphorylated, as
determined by phosphoamino acid analysis (data not shown).
To localize the domain of the Taxol-induced serine phosphorylation, the
32P-labeled Shc proteins obtained by
immunoprecipitation with anti-Shc antibody were resolved by SDS-PAGE
and transferred to nitrocellulose, and the region of the blot
containing p66shc was digested with CNBr in 70% formic acid.
Approximately 70–90% of the 32P-labeled p66shc
was released from the nitrocellulose after CNBr treatment.
SDS-PAGE/autoradiography of the CNBr-released peptides from p66shc of
control cells revealed the presence of a
32P-labeled fragment with an apparent molecular
weight of 7.9 kDa (Fig. 5D)
. An additional
32P-labeled band at 9.6 kDa, which most likely
represents a further phosphorylated form of the 7.9 kDa fragment,
was seen in the Taxol-treated samples. Minor labeling of 14–16-kDa
peptides also was observed that may represent incomplete digested
products of the CNBr reaction.
MEK Inhibitor PD98059 Does Not Influence the Taxol-induced
Electrophoretic Mobility Shift of p66shc and Raf-1.
It has been demonstrated in CHO cells expressing the human EGFR that
EGF induced serine/threonine phosphorylation of 50% of the 66-kDa
Shc proteins. Similar results were obtained with insulin. Both of these
effects were blocked by PD98059, a MEK-specific inhibitor, probably
through a feedback mechanism (37
, 38)
. In A549 cells, as
expected, PD98059 inhibited the EGF-stimulated electrophoretic mobility
shift of both p66shc and Raf-1 (Fig. 6
, left panels). However, PD98059 did not inhibit the
Taxol-induced mobility shift of either p66shc or Raf-1 (Fig. 6
,
right panel), suggesting that posttranslational
modifications of p66shc and Raf-1 mediated by Taxol do not involve
activation of MEK.
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. A Taxol-induced shift was observed in A549-T12 cells only at a
significantly higher concentration of drug, such as 200-1000
nM, although the extent of shift even at these
concentrations was diminished, compared with the sensitive cell line.
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, incubation of A549 cells with these drugs (16 h, 50 nM)
led to a p66shc mobility shift and phosphorylation of Raf-1. Baccatin,
an inactive Taxol analogue that, at 50 nM, does not
stabilize microtubules or block cells in mitosis, did not induce these
effects. The same results as those seen with baccatin were observed
with Adriamycin (0.3 µM), methrotrexate (1
µM), etoposide (5 µM), and 5-fluorouracil
(10 µM), all cytotoxic compounds that do not interact
with the tubulin/microtubule system and are not antimitotic agents.
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DISCUSSION |
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Inspection of the deduced amino acid sequence of human p66shc
(36)
revealed that there were only three predicted CNBr
fragments with masses in the 6–8-kDa range. These correspond to
residues 2–65 (6683 Da), 177–230 (5841 Da), and 258–329 (7957 Da).
On the basis of the following premises, it is predicted that the
7.9-kDa CNBr fragment represents residues 2–65: (a)
p52shc does not shift upon Taxol treatment. Residues 177–230 and
258–329 are shared by both p52shc and p66shc, whereas residues 2–65
are in the unique CH2 domain of p66shc. Highly charged peptides may run
anomalously on SDS-PAGE, and therefore the fragment with a 6683-Da mass
may migrate in the 7.9-kDa mass range; (b) a recent report
indicated that, in mouse embryo fibroblast cells transfected with
p66shc, alanine substitution of serine 36, in the CH2 domain, resulted
in the loss of the
H2O2-induced gel mobility
shift of p66shc and a significant reduction of serine phosphorylation
upon H2O2 treatment
(39)
; and (c) residues 2–65 contain 11
serines. Because a gradual shift of p66shc was observed as the
concentration of Taxol increased, the 9.6-kDa band is most probably
derived from the 7.9 kDa following phosphorylation after Taxol
treatment. It is interesting to note that residues 2–65 are a
proline-rich region and suggest that a proline-directed kinase may be
responsible for the phosphorylation of serine in this domain of p66shc.
Several lines of evidence indicate that the effects of Taxol on Shc in non-macrophage cells are clearly distinguishable from those effects elicited by mitogens, such as EGF: (a) Taxol does not cause tyrosine phosphorylation of Shc and does not induce Shc/Grb2 complex formation, suggesting that Ras activation is not involved; (b) the time required to induce the electrophoretic shift of p66shc by Taxol is much longer than that for EGF; and (c) the MEK inhibitor PD98059 does not inhibit the Taxol-induced p66shc mobility shift. Our results indicate that this Taxol-induced signal transduction pathway differs from classical mitogen-activated signal transduction cascades. Consistent with these findings, it has been reported that domains unique to Shc isoforms, CH1 and CH2, may be involved in a network of protein-protein interactions, and Shc may have other roles in addition to its involvement in Ras activation (28) . Because p66shc contains an extra CH2 region that is not present in p52shc, it may have unique functions compared with other Shc isoforms. In fact, it has been shown that insulin stimulates the phosphorylation of the 66- and 52-kDa Shc isoforms by distinct pathways (38) . Opposite effects of these two isoforms on the EGFR-MAP kinase-fos signaling pathway also has been reported (36) . Although EGF also caused serine/threonine phosphorylation of 50% of p66shc in CHO cells expressing human EGF receptor, unlike Taxol-induced serine phosphorylation of p66shc, this phosphorylation event occurred subsequent to tyrosine phosphorylation and was a fast response (1–3 min) prevented by pretreatment of cells with PD98059 (37) . A recent report described the role of phosphorylation at serine 36 in the CH2 domain of p66shc in the regulation of stress apoptotic response (39) .
The effect of Taxol on signaling molecules in lung carcinoma A549 cells also differs from those effects observed in murine macrophages. In mouse macrophages, high concentrations of Taxol (e.g., 10 µM) were shown to mimic LPS activities in stimulating signaling pathways and gene expression. In contrast, other microtubule-interacting agents, such as Taxotere and epothilones that promote polymerization of stable microtubules, were not able to elicit LPS-mimetic activity in murine macrophages (14 , 49 , 50) . Our results indicating that tyrosine phosphorylation is not involved in Taxol-induced signaling in A549 cells are consistent with findings from other studies in which Taxol-mediated Raf-1 and Bcl-2 hyperphosphorylation was Lck-independent in a Lck-deficient clone from a T-cell leukemia cell line, J.CAM1.6 (44) . Lck is a tyrosine kinase that is important for T-cell signaling from the plasma membrane to Raf-1 kinase (51) .
Taxol can cause mitotic arrest and apoptotic cell death. However, it is
not clear whether Taxol-induced apoptosis is a consequence of mitotic
arrest through its interactions with the tubulin/microtubule system or
is an independent event occurring via mechanisms unrelated to cell
cycle arrest. Possible mechanisms of Taxol-induced apoptosis have been
proposed (52)
. In A549 cells, it has been proposed that
Taxol-mediated cell death may result from two different mechanisms; at
low Taxol concentrations (<9 nM), cell death may occur
without a G2-M block, whereas at higher
concentrations (>9 nM), cell death may result from
terminal mitotic arrest (24)
. In this study, we
demonstrated that Taxol, at concentrations >10–20 nM,
induced p66shc serine phosphorylation that coincided with Taxol-induced
activation of Raf-1 and ERK1/2 and with Taxol-induced release of PARP
cleavage products, an early event in apoptosis. Two lines of evidence
suggest that Taxol-mediated serine phosphorylation of p66shc is related
to the blockade of cells in mitosis: (a) the gel mobility
shift of p66shc is induced not only by Taxol but also by other
microtubule-interacting agents, all of which are antimitotic drugs; and
(b) the maximum effect of the electrophoretic mobility shift
of p66shc was seen at 9–18 h after Taxol treatment, a time at which a
high proportion of the cells would be in mitosis (see Fig. 3
). The
recent finding that disruption of p66shc function, by mutating the
serine phosphorylation site, enhances cellular resistance to apoptosis
(39)
suggests that the phosphorylation of p66shc reported
here may modulate Taxol-induced apoptosis.
In a classical Ras-dependent signal transduction pathway that leads to the activation of ERK1/2, tyrosine phosphorylation of Shc results in Shc/Grb2/SOS ternary complex formation and the subsequent activation of Ras and Raf-1. In Taxol-treated A549 cells, although a Taxol-induced Raf-1 mobility shift coincides with phosphorylation of p66shc, Raf-1 phosphorylation may not be the downstream event of p66shc phosphorylation. Rather, both of these events may be the consequence of mitotic arrest resulting from the action of antimitotic agents.
The emergence of drug resistance is a significant clinical problem that develops during the treatment of most human malignancies with antitumor agents, including Taxol. Potential mechanisms involved in Taxol resistance include overexpression of P-glycoprotein (53) , mutations in tubulin that may influence its interaction with Taxol (54) , and overexpression of c-erbB-2/neu in breast cancer cells (55) . In Taxol-resistant A549-T12 cells that do not express P-glycoprotein, both the phosphorylation of p66shc and Raf-1 induced by Taxol were diminished, compared with drug-sensitive cells. The report that p66shc knockout mice have an increased resistance to oxidative stress and a longer life span (39) agrees with the results obtained with A549-T12 cells that are resistant to Taxol and whose p66shc is not activated by 20 nM Taxol.
In summary, we have identified a Taxol-mediated signaling event that is distinct from those effects elicited by micromolar concentrations of Taxol in mouse macrophages. It is also different from mitogen-activated processes, in which both tyrosine and serine/threonine phosphorylation of Shc proteins occurs rapidly. It is known that serine phosphorylation occurs during mitosis (56) , and it is probable that serine kinases are up-regulated or activated at this phase of the cell cycle. The altered serine phosphorylation of p66shc that occurs in the presence of a variety of molecules that interact with the tubulin/microtubule system may relate to the dramatically prolonged and abnormal time that such cells are in the mitotic phase of the cell cycle.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This research was supported in part by USPHS
Grant CA83185. 
2 To whom requests for reprints should be
addressed, at Department of Molecular Pharmacology, Albert Einstein
College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Phone:
(718) 430-2163; Fax: (718) 430-8922; E-mail: shorwitz{at}aecom.yu.edu
3 The abbreviations used are: MAP,
mitogen-activated protein; LPS, lipopolysaccharide; ERK, extracellular
signal-regulated kinase; MEK, mitogen activated and extracellular
signal-regulated protein kinase kinase; PARP, poly(ADP-ribose)
polymerase; EGF, epidermal growth factor; EGFR, epidermal growth factor
receptor; SOS, son of sevenless; SH2, Src homology 2; CH, collagen
homology; PP2A, protein phosphatase 2A.
Received 2/18/00. Accepted 7/19/00.
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 W. W. Smith, D. D. Norton, M. Gorospe, H. Jiang, S. Nemoto, N. J. Holbrook, T. Finkel, and J. W. Kusiak Phosphorylation of p66Shc and forkhead proteins mediates A{beta} toxicity J. Cell Biol., April 25, 2005; 169(2): 331 - 339. [Abstract] [Full Text] [PDF]
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S. Lee, W. Yang, K.-H. Lan, S. Sellappan, K. Klos, G. Hortobagyi, M.-C. Hung, and D. Yu Enhanced Sensitization to Taxol-induced Apoptosis by Herceptin Pretreatment in ErbB2-overexpressing Breast Cancer Cells Cancer Res., October 15, 2002; 62(20): 5703 - 5710. [Abstract] [Full Text] [PDF]
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 H. M. McDaid and S. B. Horwitz Selective Potentiation of Paclitaxel (Taxol)-Induced Cell Death by Mitogen-Activated Protein Kinase Kinase Inhibition in Human Cancer Cell Lines Mol. Pharmacol., August 1, 2001; 60(2): 290 - 301. [Abstract] [Full Text] [PDF]
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J.-i. Okano and A. K. Rustgi Paclitaxel Induces Prolonged Activation of the Ras/MEK/ERK Pathway Independently of Activating the Programmed Cell Death Machinery J. Biol. Chem., May 25, 2001; 276(22): 19555 - 19564. [Abstract] [Full Text] [PDF]
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| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
