| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Department of Radiation Oncology, Division of Radiation and Cancer Biology, University of Michigan Medical School, University of Michigan Comprehensive Cancer Center, Ann Arbor, Michigan 48109-0948
Address all correspondence and requests for reprints to: Stephen P. Ethier, Ph.D., 1500 East Medical Center Drive, Ann Arbor, Michigan 48109-0948. E-mail: spethier{at}umich.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Work in our laboratory over the past several years has focused on understanding the physiological consequences of erbB-2 overexpression in human breast epithelial cells. In particular, we have attempted to understand which altered growth phenotypes of human breast cancer cells can be specifically mediated by erbB-2 when it is overexpressed and activated. To approach this question, we first studied the biological activity of the HRGs, which are potent activators of erbB-2. It is now known that HRG does not bind directly to erbB-2 but, rather, activates erbB-2 when present as a heterodimer with erbB-3 and, to a lesser extent, with epidermal growth factor receptor (EGFR) and erbB-4 (19, 20, 24, 27, 28). We demonstrated that HRG has the unique property of functioning as both an insulin-like growth factor (IGF)-like and EGF-like mitogen in mammary epithelial cells (21). Thus, whereas normal human mammary epithelial (HME) cells have a strict dependence on the synergistic interaction between EGF and IGF-I for growth under serum-free conditions (29), HRG (particularly HRG-ß) can substitute for either growth factor to stimulate rapid growth of HME cells. This property is not shared by any other members of the EGF or IGF family of growth factors. It seems that activation of erbB-2/erbB-3 heterodimers is responsible for the dual specific nature of the HRG response. Docking molecules that bind activated erbB-2 stimulate the mitogen-activated protein (MAP) kinase pathway, thereby sending an EGFR-like signal (23); whereas, tyrosine phosphorylated erbB-3 is a potent activator of the phosphatidyl inositol (PI) 3'-kinase pathway (30, 31, 32), which is activated by the IGF-I receptor under normal conditions (33, 34, 35, 36, 37, 38).
Based on these observations, we hypothesized that human breast cancer cells with amplified and overexpressed erbB-2 would become independent of IGF-I and EGF for growth under serum-free conditions. Experiments using the 21T series of human breast cancer cell lines were consistent with this hypothesis. 21MT-2 cells express intermediate erbB-2 levels and are IGF-I independent for growth, whereas 21MT1 cells, which express very high levels of activated erbB-2, are independent of both IGF-I and EGF for continuous growth in serum-free medium (39).
The growth factor independence expressed by the 21T human breast cancer cells was consistent with predictions made from the biological activity of erbB-2/3 after HRG stimulation. However, these breast cancer cells contain many genetic alterations that could cooperate with, or act independently of, erbB-2 to mediate these altered growth phenotypes. Therefore, the purpose of the present studies was to determine whether transduction of human erbB-2 into two normal HME cell lines, which were immortalized by different mechanisms, results in growth factor-independent proliferation when the gene is expressed at levels comparable with breast cancer cells with an erbB-2 gene amplification. The data indicate that transduction of erbB-2, followed by long-term selection in growth factor-deficient medium, results in the emergence of growth factor-independent cells that dramatically overexpress p185erbB-2. However, the degree of constitutive activation of p185erbB-2 and the extent of growth factor independence in the two cell lines differed. The H16N2 cell line, which was immortalized using the HPV-16 genome, expressed higher levels of tyrosine phosphorylated p185erbB-2 than MCF-10A cells, which were spontaneously immortalized in culture. In addition, H16N2-erbB-2 cells were independent of both insulin and EGF, whereas MCF-10A-erbB-2 cells maintained their EGF-dependency for growth. Thus, overexpression and constitutive activation of erbB-2 can, by itself, induce multiple growth factor-independent proliferation of normal HME cells. However, cellular factors, possibly involving HPV-16-mediated disruption of the Rb and p53 pathways, influence the levels of constitutive receptor activation and, in turn, the degree of growth factor independence of the cells.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
erbB-2 cloning and virus preparation
The full-length human erbB-2 gene was subcloned in the
XhoI site in the pTP2000 retroviral vector. Five micrograms
of DNA was transfected in ØNX-A cells. Forty-eight hours later,
conditioned medium was collected, cell debris was pelleted, the
supernatant was passed through a 0.45-µm syringe filter, and the
virus was frozen at -80 C. Virus was thawed and allowed to infect
MCF-10A and H16N2 cells in the presence of polybrene for 24 h.
Membrane preparations and protein blots
Cells were dounced in a buffer consisting of 20
mM HEPES (pH 7.4), 5 mM
Na3VO4, 1 mM
phenylmethylsulfonylflouride, and 10 mM pyrophosphate.
Membrane fractions were retrieved by centrifuging at 100,000 x
g for 35 min at 4 C. Membrane fractions were resuspended in
lysis buffer (10 mM sodium phosphate (pH 7.5), 100
mM NaCl, 1% Triton X-100, 0.5%
Na+deoxycholate, and 0.1% SDS). Protein concentrations
were equalized using the Løwry method (Bradford, 1976 no. 878).
Laemmeli sample buffer (Sambrook, 1989 no. 42) was added, and the
samples were boiled. Equal amounts of protein were separated in 7.5%
SDS-PAGE. Proteins were blotted to polyvinylidene diflouride (PVDF)
membrane and probed with either 
-erbB-2 (clone 9.3),
-Ptyr4G10 (catalog no. 05–321, Upstate Biotechnology, Inc., Lake Placid, NY), or -erbB-3 (catalog
no. PC27, Oncogene Research Products, Cambridge, MA) antibodies.
Flow cytometry analysis
Infected cells were washed three times in PBS, and 1 µg/ml
anti-erbB-2 antibody (clone 254) was incubated with the cells for
1 h at room temperature. Cells were then incubated with secondary
antibody for 1 h at room temperature, then with fluorescein avidin
DCS in 0.1 M NaHCO3 buffered saline (pH 8.2) for 30 min at
room temperature. Cells were removed from the tissue culture dish with
10 mM EDTA and fixed with 95% ethanol. Cells that were
sterily sorted were not fixed.
Immunocytochemistry
Rinsed cells were fixed in 100% methanol at -20 C for 20 min.
Cells were incubated with 1 µg/ml erbB-2 antibody (clone 254) for 30
min at room temperature, then with biotintylated secondary antibody for
30 min at room temperature. Cell-surface erbB-2 was visualized using
the Vectastain ABC kit (catalog no. PK-4000, Vector Laboratories, Inc., Burlingame, CA).
Southern blot
Ten micrograms of genomic DNA was digested with KpnI.
The restriction fragments were separated on an agarose gel, transferred
by standard methods to nylon membrane, probed with radioactive erbB-2
complementary DNA (cDNA), quantitated using a Molecular Dynamics Storm
phosphorimager (Molecular Dynamics, Inc., Sunnyvale, CA),
and visualized by autoradiography.
Northern blot
Ten micrograms of total cellular RNA was separated on a
formaldehyde-containing agarose gel and transferred to nylon membrane
by standard methods. Blots were probed with radioactive erbB-2 cDNA and
visualized by autoradiography.
Immunoprecipitation
Cells were lysed in a buffer containing 20 mM
Tris·HCl (pH 8.0), 137 mM NaCl, 1% NP-40, 10% glycerol,
1 mM Na3VO4, 1 mM
phenylmethylsulfonylflouride, 1% aprotinin, and 20 µg/ml leupeptin.
Protein concentrations were equalized using the Løwry method
(Bradford, 1976 no. 878). Laemmeli sample buffer (Sambrook, 1989 no.
42) was added, and the samples were boiled. Equal amounts of protein
were separated in 7.5% SDS-PAGE. Proteins were blotted to PVDF
membrane and probed with -Ptyr4G10 (catalog no. 05–321,
Upstate Biotechnology, Inc.).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
shows the general features of the
bicistronic retroviral vector used in these studies.
|
). Examination
of the cells by flow cytometry (not shown) and immunocytochemistry
demonstrated that, despite the use of the bicistronic vector, only
about half of the MCF-10A cells stained positively for erbB-2 (Fig. 1C, left panels).
Next, experiments were performed to determine whether the moderate
level of erbB-2 overexpression observed in infected MCF-10A cells
resulted in cell proliferation in the absence of individual growth
factors. MCF-10A cells that had been selected in G418-containing medium
with a full complement of growth factors were switched to insulin-free
medium and grown to confluence. Although these cells grew slowly,
erbB-2-transduced MCF-10A cells did grow continuously and were
subculturable. Reanalysis of erbB-2 expression, after selection in
insulin-free medium, demonstrated that 100% of the cells stained
positively for erbB-2 (Fig. 1C, right panels). This
selection for the erbB-2-expressing subpopulation resulted in higher
apparent levels of erbB-2, as detected in Western blots, as compared
with cells selected with G418 only (Fig. 1D). These results are
consistent with the hypothesis that erbB-2 overexpression directly
mediates IGF-independent proliferation of HME cells. However, MCF-10A
cells expressing these moderate levels of p185erbB-2 were
unable to grow in EGF-free medium.
To compare the erbB-2 expression levels of the MCF-10A cells described
above, with breast cancer cells having an erbB-2 gene amplification,
Western blot analyses were carried out using varying amounts of
membrane protein obtained from two breast cancer cell lines with an
erbB-2 amplification. The 21MT-1 cell line was developed by Band and
co-workers (41), and the SUM-190 cell line was recently developed in
our laboratory. The data in Fig. 1D show that, despite the erbB-2
overexpression exhibited by the transduced MCF-10A cells (relative to
parental cells), the overall levels of erbB-2 protein in these cells
was dramatically lower than that observed in the two breast cancer cell
lines with an erbB-2 gene amplification.
Expression of erbB-2 in retrovirally infected H16N2 cells
Similar experiments were then performed using a different
immortalized HME cell line. H16N2 cells were derived by HPV-16
infection of normal HME cell cultures (41). These immortalized cells
express luminal cytokeratins and are EGF-dependent for growth in
serum-free medium but are only marginally dependent on exogenous
insulin. The relative IGF-independence of these cells is likely the
result of their immortalization with HPV-16. The data in Fig. 2 show that, as in MCF-10A cells,
infection of H16N2 cells with pTPerbB-2 resulted in moderate
overexpression of p185erbB-2 relative to controls. In an
attempt to increase the number of cells infected with the viral vector,
H16N2 cells were infected three times, over a 12-h period, with
pTPerbB-2 followed by selection in G418-containing medium. These triply
infected cells expressed marginally higher levels of
p185erbB-2 than cells infected singly with the vector.
|
shows the erbB-2 expression profile of the
transduced H16N2 cells and indicates the fractions that were sorted.
Figure 2C shows that expression of p185erbB-2 in the
flow-sorted cells was higher than in the unsorted population of triply
infected cells. However, these cells still expressed substantially
lower levels of p185erbB-2 than breast cancer cells with an
erbB-2 gene amplification. And, like MCF-10A cells described above,
these H16N2 cells were also unable to grow in EGF-free medium. These experiments demonstrate that infection of two different HME cell lines with the pTPerbB-2 expression vector, followed by short-term selection in G418-containing medium, results in cells that overexpress p185erbB-2 (relative to the parental cells) but express lower levels than breast cancer cells with an erbB-2 amplification. Furthermore, these transduced HME cells can grow in the absence of insulin but not in the absence of EGF.
Generation of HME cells with high-level erbB-2
overexpression
Experiments were performed to determine whether direct, long-term
selection of pTPerbB-2-infected cells for growth factor independence
would yield cell populations expressing high levels of
p185erbB-2. MCF-10A or H16N2 cells were infected with
pTPerbB-2 or pTPneo and switched, 24 h later, to G418-containing
medium without insulin or without insulin and EGF. Switching to growth
factor-deficient medium, in this way, dramatically reduced the
proliferative capacity of both cell lines, regardless of which vector
was used to infect the cells. However, cells infected with pTPerbB-2
(but not control virus) and cultured continuously in growth factor
deficient-medium slowly grew to confluence and could be subcultured.
For both lines, cells had to be maintained for several weeks under
these culture conditions before they became confluent. However, once
confluent, cells in subsequent passages grew increasingly rapidly until
achieving a growth rate comparable with control cells in the presence
of growth factors. Thus, long-term selection in growth factor-deficient
medium did not result in the prompt emergence of growth
factor-independent clones but, rather, resulted in the slow adaptation
of the entire population of G418-resistant cells to these conditions.
Western blot analysis of erbB-2- transduced growth factor-independent
cells, compared with breast cancer cells with an erbB-2 gene
amplification, is shown in Fig. 3A. These
data demonstrate that growth factor-independent MCF-10erbB-2 and
H16N2erbB-2 cells that emerged after long-term selection in growth
factor-deficient medium expressed p185erbB-2 levels that
were equivalent to that expressed by breast cancer cells with an erbB-2
gene amplification. Phosphotyrosine Western blot analysis of membrane
protein obtained from these cells indicated that growth
factor-independent cells expressed high levels of constitutively
tyrosine-phosphorylated protein in the 185-kDa range (Fig. 3B).
Immunoprecipitation, using erbB-2 specific antibodies, followed by
phosphotyrosine Western blotting, demonstrated that
p185erbB-2 was indeed highly tyrosine phosphorylated in
these cells (Fig. 3C). In addition, a tyrosine-phosphorylated p170 was
coimmunoprecipitated with erbB-2, which was shown to be the EGF
receptor by probing the blots with an EGF receptor-specific antibody
(not shown). These data further support the hypothesis of a direct
relationship between the level of erbB-2 expression and growth
factor-independent proliferation of HME cells.
|
, B and C, show
that the levels of tyrosine phosphorylated p185erbB-2 in
the MCF-10AerbB-2 cells were lower than in either the H16N2erbB-2 cells
or the SUM-190 cells. This difference could account for the continued
EGF-dependence of the MCF-10AerbB-2 cells. The high level expression
and constitutive activation of p185erbB-2 found in
H16N2-erbB-2 cells, relative to MCF-10A-erbB-2 cells, may reflect
inherent differences between the two cell lines or may be the result of
alterations induced by the HPV-16 genome that was used to immortalize
the H16N2 cells. Further work will be required to explore this
possibility.
Constitutive activation of erbB-3 in erbB-2-overexpressing HME
cells
Because the growth factor requirements of the HME cell lines were
altered by progressive overexpression of erbB-2, experiments to examine
the erbB-3 expression and activation status of these cells were
performed. Phosphotyrosine Western analysis of erbB-3
immunoprecipitates, derived from the different erbB-2-transduced cell
lines and controls, demonstrated that p180erbB-3 became
activated to increasingly high levels in the growth factor-independent
cells. Indeed, the H16N2erbB-2 cells that were both insulin- and
EGF-independent, expressed the highest levels of
tyrosine-phosphorylated erbB-3 (Fig. 4A).
Under the conditions used for erbB-3 immunoprecipitation, erbB-2 was
not coimmunoprecipitated (not shown). Probing of Western blots of
erbB-3 immunoprecipitates with erbB-3 antibodies demonstrated that
overall levels of p180erbB-3 were not changed in the
erbB-2-overexpressing cells (Fig. 4B). Thus, progressive overexpression
of erbB-2 in HME cells results in the constitutive activation of erbB-3
without changes in the level of p180erbB-3. Furthermore,
the level of activation of both erbB-2 and erbB-3 correlates with
progressive growth factor independence.
|
demonstrate
that long-term selection of cells, either singly- or triply-infected
with pTPerbB-2, did not result in increases in erbB-2 insert number
relative to cells that were transduced and directly flow sorted. By
contrast, erbB-2 messenger RNA levels were dramatically increased in
the multiple growth factor-independent cells.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The situation in human breast cancer cells is analogous to what was seen in rat mammary carcinoma cells, but the mechanism of erbB-2 activation seems different, at least in the setting of gene amplification. Human breast cancer cells with an erbB-2 gene amplification dramatically overexpress the protein, relative to normal HME cells (1, 8, 45). Furthermore, erbB-2 protein is constitutively tyrosine phosphorylated in the absence of any demonstrable autocrine growth factor activity (39). Thus, in this case, constitutive activation of p185erbB-2 seems to be the direct result of the overexpression of the protein, as has been suggested by others (46). In addition, breast cancer cells were found to be independent of IGF-I for growth when erbB-2 was amplified and overexpressed to moderate levels and both IGF-I- and EGF-independent when erbB-2 was overexpressed to very high levels.
In the present studies, we found that transduction of full-length erbB-2 into immortalized HME cells results in erbB-2 overexpression. However, without long-term selection in the absence of growth factors, the pTPerbB-2 vector induced only moderate increases in p185erbB-2 expression, relative to parental control cells and these levels were not sufficient for rapid growth in the absence of insulin or EGF. When maintained in insulin-free, or insulin and EGF-free medium, MCF-10A cells and H16N2 cells slowly expanded and, after being subcultured, grew increasingly well in the absence of growth factors. The growth factor-independent cells, so derived, expressed very high levels of erbB-2 protein, which were equivalent to that expressed by breast cancer cells with an erbB-2 gene amplification. The high-level erbB-2 expression was not the result of an increase in insert copy number but, rather, occurred at the transcriptional level. Furthermore, erbB-2 and erbB-3 expressed by these growth factor-independent cells were constitutively tyrosine phosphorylated, just as we had observed previously with breast cancer cells.
Despite the high-level erbB-2 expression obtained in these HME cells, only the H16N2 cells became independent of both insulin and EGF. MCF-10AerbB-2 cells, although insulin-independent for growth, still require exogenous EGF. The levels of p185erbB-2 expressed by the MCF-10AerbB-2 cells are at least as high as the levels found in the SUM-190 breast cancer cells, which are independent of both insulin and EGF. This indicates that overexpression of erbB-2 is sufficient to mediate growth of HME cells in the absence of IGFs but not sufficient to make cells EGF independent. The observation that both 21MT-1 cells and SUM-190PT cells are independent of both insulin and EGF suggests that erbB-2 overexpression activates signaling pathways that act in concert with other alterations present in breast cancer cells, to yield EGF-independence. That H16N2erbB-2 cells are also EGF-independent suggests that specific changes mediated by the HPV-16 E5, E6, or E7 proteins cooperate with erbB-2 overexpression to complete the transformation to EGF-independence. Studies are currently underway to test this hypothesis.
The ability of HRG to act as an IGF-like mitogen, and for constitutive erbB-2 activation to result in IGF-I-independent growth of mammary epithelial cells, is a consistent and intriguing observation in our laboratory. The potent activation of the PI 3'-kinase pathway by erbB-3 and (to a lesser extent) erbB-2 may explain this important aspect of erbB-2/erbB-3 signal transduction (31, 32). The cytoplasmic domain of erbB-3 differs significantly from that of the other members of the erbB family, by having six YXXM motifs, which serve as docking sites for the p85 regulatory subunit of PI 3'-kinase. Therefore, erbB-3 is a potent activator of this enzyme. We have demonstrated high-level PI 3'-kinase activity in growth factor-independent, erbB-2-overexpressing human breast cancer cells and in HME cells stimulated with HRG (30). This is important because, in normal HME cells, which require both IGF-I and EGF for growth, activation of the PI 3'-kinase pathway is driven predominantly by IGF-I signaling (34, 47, 48). Activation of the IGF-I receptor results in tyrosine phosphorylation of IRS-I, which contains 20 YXXM sites for PI 3'-kinase activation. Site-specific mutagenesis studies have demonstrated that the mitogenic activity of the IGF-I receptor is dependent on the integrity of the binding sites for p85 (33). Thus, the synergistic interaction between IGF-I and EGF, seen for most epithelial cell types, is the result of complementary signaling pathways activated by the two mitogens; the ras/raf/MAP kinase pathway by EGF, and the PI 3'-kinase pathway by IGF-I. The observation that progressive overexpression and activation of erbB-2 in our cells yields a progressive increase in the levels of erbB-3 tyrosine phosphorylation without changes in erbB-3 protein levels is consistent with the hypothesis that erbB-2 and erbB-3 interact to drive altered phenotypes of breast cancer cells.
The ability of activated erbB-2 to stimulate both PI 3'-kinase and the MAP kinase pathways (23) has profound implications for its behavior as an oncoprotein. Several groups have demonstrated the importance of the PI 3'-kinase pathway as a mediator of cell survival and apoptosis resistance (37, 38, 49, 50, 51, 52, 53). Thus, many cell types can be induced to undergo programmed cell death by blocking signals either from the IGF-I receptor, from PI 3'-kinase itself, or from the AKT kinase that is activated by PI 3'-kinase. In addition, the finding that the proapoptotic protein BAD can be phosphorylated by AKT, effectively preventing its interaction with BCL-2, provides mechanistic support for the hypothesis that PI 3'-kinase signaling is important for cell survival (54, 55).
Taken together, these findings suggest that erbB-2 is a potent oncogene, because it not only has the ability to drive cell proliferation but also has the ability to signal in ways that prevent programmed cell death. The results obtained from our work support this model and demonstrate directly that overexpression of erbB-2 results in the constitutive activation of both p185erbB-2 and erbB-3, which in turn, results in independence of IGF-I and, in some cases, EGF.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received October 1, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  J. P. Gustin, B. Karakas, M. B. Weiss, A. M. Abukhdeir, J. Lauring, J. P. Garay, D. Cosgrove, A. Tamaki, H. Konishi, Y. Konishi, et al. Knockin of mutant PIK3CA activates multiple oncogenic pathways PNAS, February 24, 2009; 106(8): 2835 - 2840. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. H. Tomasson, Z. Xiang, R. Walgren, Y. Zhao, Y. Kasai, T. Miner, R. E. Ries, O. Lubman, D. H. Fremont, M. D. McLellan, et al. Somatic mutations and germline sequence variants in the expressed tyrosine kinase genes of patients with de novo acute myeloid leukemia Blood, May 1, 2008; 111(9): 4797 - 4808. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. M. Diehl, E. T. Keller, and K. M. Woods Ignatoski Why should we still care about oncogenes? Mol. Cancer Ther., February 1, 2007; 6(2): 418 - 427. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Q. Yang, K. L. Streicher, M. E. Ray, J. Abrams, and S. P. Ethier Multiple Interacting Oncogenes on the 8p11-p12 Amplicon in Human Breast Cancer Cancer Res., December 15, 2006; 66(24): 11632 - 11643. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Haslekas, K. Breen, K. W. Pedersen, L. E. Johannessen, E. Stang, and I. H. Madshus The Inhibitory Effect of ErbB2 on Epidermal Growth Factor-induced Formation of Clathrin-coated Pits Correlates with Retention of Epidermal Growth Factor Receptor-ErbB2 Oligomeric Complexes at the Plasma Membrane Mol. Biol. Cell, December 1, 2005; 16(12): 5832 - 5842. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. L. Morse, H. Gray, C. M. Payne, and R. J. Gillies Docetaxel induces cell death through mitotic catastrophe in human breast cancer cells Mol. Cancer Ther., October 1, 2005; 4(10): 1495 - 1504. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. B. Moffa, S. L. Tannheimer, and S. P. Ethier Transforming Potential of Alternatively Spliced Variants of Fibroblast Growth Factor Receptor 2 in Human Mammary Epithelial Cells Mol. Cancer Res., November 1, 2004; 2(11): 643 - 652. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. G. Kleer, Q. Cao, S. Varambally, R. Shen, I. Ota, S. A. Tomlins, D. Ghosh, R. G. A. B. Sewalt, A. P. Otte, D. F. Hayes, et al. EZH2 is a marker of aggressive breast cancer and promotes neoplastic transformation of breast epithelial cells PNAS, September 30, 2003; 100(20): 11606 - 11611. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. M. Woods Ignatoski, D. L. Livant, S. Markwart, N. K. Grewal, and S. P. Ethier The Role of Phosphatidylinositol 3'-Kinase and Its Downstream Signals in erbB-2-Mediated Transformation Mol. Cancer Res., May 1, 2003; 1(7): 551 - 560. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. S. Hendriks, L. K. Opresko, H. S. Wiley, and D. Lauffenburger Coregulation of Epidermal Growth Factor Receptor/Human Epidermal Growth Factor Receptor 2 (HER2) Levels and Locations: Quantitative Analysis of HER2 Overexpression Effects Cancer Res., March 1, 2003; 63(5): 1130 - 1137. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Kumar-Sinha, K. W. Ignatoski, M. E. Lippman, S. P. Ethier, and A. M. Chinnaiyan Transcriptome Analysis of HER2 Reveals a Molecular Connection to Fatty Acid Synthesis Cancer Res., January 1, 2003; 63(1): 132 - 139. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
