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Department of Surgery, Washington University School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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However, monoclonal antibody targeting of p185 appears to result in primarily cytostatic, as opposed to cytotoxic, effects on susceptible tumor cells (9 , 12) . Furthermore, tumor cells that overexpress p185 can shed soluble antigenic fragments from the cell membrane, which may interfere with monoclonal antibodies reaching the tumor cell surface in vitro (13) and in vivo (10) . These limitations have led to the development of a number of distinct approaches to inhibiting p185 expression or function, including the use of chemical inhibitors of p185 tyrosine kinase activity (14) , viral mechanisms of inhibiting HER2/neu expression (15) , gene transfer of single chain antibody (16) or dominant negative HER2/neu mutants (17) and treatment with antisense oligonucleotides (18, 19, 20, 21) . Here we describe the effects of a phosphorothioate antisense oligodeoxyribonucleotide directed against the 5' region of the HER2/neu mRNA molecule on HER2/neu expression, cell proliferation, and apoptotic cell death in HER2/neu-overexpressing cancer cells derived from tumors of several tissue types.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Oligodeoxynucleotide Treatment.
Phosphorothioate
ODNs3
targeting the 5' region of the HER2/neu mRNA molecule were obtained
from Oligos, Etc (Wilsonville, OR), bases in bold font indicate
substitutions:
Antisense: CTCCATGGTGCTCAC
Sense: GTGAGCACCATGGAG
Scrambled antisense: CGCCTTATCCGTAGC
One-mismatch: CTCCATGCTGCTCAC
Four-mismatch: CTACCTGCTGCTGAC
The lyophilized ODNs were reconstituted in sterile distilled water to 1 mM, filter-sterilized, and stored in aliquots at -20°C as stock solutions. For subsequent experiments, the stock solutions of ODNs were diluted to give final concentration of 1 µM. Diluted ODNs were mixed with 10 µg/ml Lipofectin (Life Technologies, Inc., Gaithersburg, MD), and cells were exposed to the mixture for 4 h, after which the mixture-containing medium was replaced with the culture medium.
Western Blotting.
Crude cell extracts were obtained by direct lysis of the cells in lysis
buffer (50 mM Tris-HCl, pH 7.4, 5 mM EGTA, 1%
Triton X-100, 150 mM NaCl, 2 mM
phenylmethylsulfonyl fluoride, 1% Aprotinin, and 5 µg/ml Leupeptin).
Twenty µg of the lysate from each of the cell line were subjected to
7.5% SDS-PAGE and electroblotted onto a PVDF membrane (Immobilon-P;
Millipore, Bedford, MA). Blots were hybridized with specific
antibodies, followed by a species-specific alkaline
phosphatase-conjugated second antibody, and were developed using the
CDP-star chemiluminescent system. In antisense experiments, cell
extracts were prepared 48 h (unless otherwise noted) after the
initiation of treatment as described for Northern blotting, and 2 µg
of lysates were separated by SDS-PAGE. Antibodies specific for
p185HER2/neu were obtained from
Oncogene Research (Cambridge, MA) and used according to the supplier’s
recommendations. Anti-PARP and anti-cyclin B antibodies were purchased
from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). An actin-specific
monoclonal antibody (Amersham Corp., Arlington Heights, IL) was used as
a control for protein loading.
Cell Growth Assay.
One-ml aliquots containing 2 x 104 cells were plated into the wells of
24-well culture plates and were allowed to adhere for 24 h. After
cells were firmly attached, they were washed once with prewarmed
OPTIMEM-I (Life Technologies, Inc.), and 10 µg/ml Lipofectin mixture
with or without 1 µM antisense, sense, or scrambled
antisense ODNs were applied to each well for 4 h at 37°C. After
the 4-h incubation, the ODN mixture was replaced with regular serum
containing culture medium, and the cells were incubated for an
additional 5 days. At the indicated times, cells were detached from the
wells with trypsin-EDTA and counted with a hemocytometer. Only viable
cells based on trypan blue dye exclusion were counted. Results are the
means of triplicate samples.
Flow-Cytometric Detection of Apoptosis.
BT474 cells (5 x 105) were
treated with 1.5 ml of ODN mixture in 60-mm culture dishes as
described. After an additional incubation of 72 h, cells were
detached from the culture dish and collected via centrifugation.
Floating cells from the supernatant were combined with the trypsinized
cells. Pooled cells were washed once with the growth medium and stained
with Annexin V-FITC and propidium iodide (ApoAlert Annexin V Apoptosis
Kit; Clontech), according to the manufacturer’s protocol. After cells
were stained in the dark for 10 min at 25°C, their fluorescence was
analyzed via flow cytometry using a FACScan (Becton Dickinson).
CPP32 Assay.
Cells (1 x 106) were treated in
100-mm culture dishes with 5 ml of ODN mixture as described previously.
After incubating for 72 h from the initiation of the treatment,
cells were detached from the culture dish and washed once with PBS.
Activation of CPP32 protease was determined by the ApoAlert CPP32 Assay
kit (Clontech), according to the manufacturer’s protocol. Absorbance
of the DEVD-pNA cleavage product pNA at 405 nm from each sample was
converted to CPP32 units using the standard curve generated by the
absorbance of known amount of pNA.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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As shown in Fig. 1A
, treatment of BT474 breast carcinoma cells with
HER2/neu-specific antisense ODNs results in dose-dependent
inhibition of p185 expression. There is no effect of control sense or
scrambled antisense ODNs on p185 expression. The biological effects of
HER2/neu antisense ODN treatment on BT474 growth parallel
the biochemical effects on p185 expression. As shown in Fig. 1B
, HER2/neu-specific ODNs have modest but
reproducible tumor inhibitory effects at concentrations as low as 30
nM, with more potent effects (>80% inhibition
of cell growth) at 1 µM. In contrast, control
sense and scrambled antisense sequences have only minimal nonspecific
effects on cell growth at concentrations 
1
µM. To avoid such nonspecific toxic effects,
all subsequent experiments have been performed using a
1-µM treatment dose.
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. There is little effect of control sense
and scrambled antisense sequences. Examination of the effects of
mismatch sequences demonstrates the stringent sequence-specificity of
the antisense effect. There is marked diminution of the activity of the
one-mismatch sequence in terms of its effect on p185 protein levels,
whereas the four-mismatch antisense sequence has essentially no effect
on p185 expression.
The effects of these sequences on p185 protein levels correlate
with their effects on BT474 tumor cell proliferation. As shown in Fig. 1D
, antisense ODNs inhibit BT474 cell growth by >80%,
whereas control ODNs have only a modest effect on cell growth,
confirming earlier results. The one-base mismatch antisense ODN has a
markedly less potent effect on BT474 cell growth, and the four-base
mismatch sequence has an effect little different from those of sense
and scramble control ODNs. Thus, the effects of antisense molecules on
p185 expression and on tumor cell proliferation appear to be critically
dependent on sequence complementarity with the HER2/neu mRNA
molecule.
HER2/neu Antisense Treatment Results in Cell Death and
Cell Surface Changes Associated with Apoptosis.
It was observed that BT474 cell culture dishes treated with
HER2/neu antisense ODNs not only contained fewer viable
tumor cells, but also contained an increased fraction of dead floating
cells compared with cultures treated with either Lipofectin alone or
with control ODNs. It was hypothesized that some of the
HER2/neu antisense effect on tumor cell growth was not
simply the result of inhibition of tumor cell proliferation, but might
actually be due to an antisense-mediated increase in apoptotic cell
death. To address this question, the fractions of cells in various
stages of apoptotic death were assessed by simultaneous Annexin V and
propidium iodide staining, using flow cytometry, 72 h after
treatment with antisense or control ODNs. A significant fraction of
BT474 tumor cells treated with HER2/neu antisense ODNs were
in the early (Fig. 2B
, lower right quadrant) or late (Fig. 2B
, upper right quadrant) stages of apoptotic
cell death. In contrast, there was little effect of sense (Fig. 2C)
or scrambled antisense (Fig. 2D)
ODNs on
apoptotic cell death, which was comparable to that seen in cells
treated with Lipofectin alone (Fig. 2A)
. Cytometric analysis
also revealed that HER2/neu antisense-treated cells showed
evidence of nuclear condensation and an increased fraction of nuclei
containing a sub-2N DNA content, an indicator of apoptotic cell death
(Table 1)
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, BT474 cells treated with HER2/neu-specific
antisense ODNs demonstrated activation of the caspase CPP32, also known
as caspase 3, whereas cells treated with control ODNs did not.
Moreover, antisense-treated BT474 cells showed evidence of degradation
of the proteins PARP and cyclin B, which are substrates of CPP32 and
other proteases activated in apoptotic cells (Fig. 3B)
.
Thus, by multiple criteria, it was demonstrated that
HER2/neu antisense ODN treatment resulted in activation of
apoptotic cell death pathways in BT474 breast carcinoma cell lines.
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. The effects on cell growth mirrored the effects of
antisense treatment in triggering apoptosis as demonstrated by CPP32
activation (Fig. 4C)
. These findings demonstrate that a
variety of tumor cell types are sensitive to the pro-apoptotic effects
resulting from antisense-mediated down-regulation of
HER2/neu expression. These results also suggest that the
growth inhibitory effects of HER2/neu-specific antisense
ODNs result in part from triggering apoptotic cell death in cells that
overexpress HER2/neu. It is of interest that the CFPAC cell line, which only moderately overexpresses p185, was quite sensitive to the effects of HER2/neu down-regulation, whereas the MCF-7 cell line, which expresses somewhat lower levels of p185, was not affected by antisense ODN exposure. Examination of the sensitivity of these cell lines to growth inhibition after p185 down-regulation by a distinct mechanism, using p185-specific monoclonal antibodies, confirmed their relative sensitivity and resistance to HER2/neu-directed therapy.4 This may reflect a threshold level of p185 overexpression required for sensitivity to HER2/neu down-regulation.
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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A number of reports of antisense effects on tumor cell proliferation have been published; in several cases it was subsequently demonstrated that non-antisense mechanisms accounted for the tumor inhibitory effects (22 , 23 , 25 , 26) . ODNs can bind growth factors (23) and their receptors in sequence non-specific fashion (25) . They may also bind other cellular proteins, resulting in aptameric effects (26) . The results presented here are unlikely to be due to such non-antisense mechanisms for a number of reasons, including avoidance of sequences known to cause non-antisense effects (23 , 25 , 26) ; the use of relatively short (15 base) ODNs at concentrations of 1 µM and below; demonstration of parallel effects of antisense compounds on protein expression as well as tumor cell growth; and the use of multiple control ODNs and tumor targets that define the effect in terms of antisense sequence and target cell susceptibility.
It is interesting to compare the results presented here with prior studies of HER2/neu down-regulation using monoclonal antibodies or antisense ODNs. Monoclonal antibodies specific for p185 have been demonstrated to have inhibitory effects on the growth of some tumor lines in cell culture (9) , including several of the lines tested here. In general, these antibodies appear to be exclusively active in inhibiting the growth of cancer cells that strongly overexpress HER2/neu, suggesting that such tumor cells are critically dependent on p185-mediated mitogenic signaling.
Antisense compounds directed against the HER2/neu oncogene have also been shown to inhibit the growth of some breast carcinoma and ovarian carcinoma cell lines (18, 19, 20, 21) . These agents also appear to be most effective against tumor cells that overexpress HER2/neu. We have demonstrated that other tumor cell types, including lung and pancreatic adenocarcinoma cell lines, can be inhibited by HER2/neu-specific antisense ODNs. Preliminary data from our laboratory suggest that antisense ODNs directed against HER2/neu sequences are superior to monoclonal antibodies specific for p185 at both down-regulating HER2/neu expression and inhibiting the in vitro growth of tumors that overexpress HER2/neu.4 Whether HER2/neu antisense ODNs are more potent than p185-specific monoclonal antibodies in vivo, and whether the combination of such agents can result in enhanced antitumor effects will require further study.
Perhaps the most intriguing finding presented here is the observation that antisense-mediated down-regulation of HER2/neu expression in tumor cell lines that overexpress HER2/neu is not simply cytostatic, but actually results in activation of apoptotic cell death mechanisms. Standard monoclonal antibodies reactive with p185 have not been shown to activate cell death pathways in tumor cells that overexpress HER2/neu (9 , 12) .4 However, multimeric antibodies directed against p185, as well as intracellular expression of a single-chain p185-specific antibody, have been shown to activate apoptotic cell death pathways in tumor cells that overexpress HER2/neu (16 , 27) . These prior reports suggested that factors specific to the altered anti-p185 antibodies, rather than the result of interfering with p185 expression, were critical in inducing apoptosis (16 , 27) . The studies presented here demonstrate that down-regulation of p185 by a non-antibody mechanism, using antisense ODNs, can also trigger cell death in cancer cells that overexpress the p185 protein. Thus, cancer cells that overexpress HER2/neu are dependent on p185 for cell survival as well as proliferation, and strategies that interfere with p185 expression or function can induce apoptotic cell death.
The molecular pathways by which HER2/neu overexpression interacts with cell death/survival signaling have not been defined. Several of the cell lines studied here, including BT474, have known p53 mutations (28) . Thus, the effects of HER2/neu-specific antisense ODNs in activating apoptosis must be p53-independent. It has been demonstrated previously that increased HER2/neu expression resulting from gene transfection can increase Bcl-2 levels (29) . Furthermore, p185 physically associates with FAS ligand on tumor cell surfaces and may interfere with FAS signaling by this mechanism (30) . Thus, it is possible that modulation of Bcl-2 and/or FAS ligand function links HER2/neu overexpression to cell survival signaling. Regardless of the underlying molecular pathways involved, the triggering of apoptotic cell death following p185 down-regulation may prove a useful approach to the therapy of tumors that overexpress HER2/neu.
HER2/neu overexpression has also been shown to play a role in resistance to the lethal effects of tumor necrosis factor (24) and chemotherapy (31 , 32) . Because these both are activators of apoptotic cell death, this may provide yet another example in which the level of HER2/neu expression contributes to altering the balance between tumor cell survival and cell death signals. We have demonstrated that antisense-mediated down-regulation of HER2/neu expression can, by itself, shift this balance in favor of tumor destruction. Antisense compounds are beginning to enter clinical trials in patients with cancer and other diseases (33, 34, 35) . The ability of HER2/neu antisense ODNs to inhibit tumor growth, using in vivo xenograft models, and to potentiate the cytotoxic effects of other modalities, such as chemotherapy, currently is under investigation (36) .
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FOOTNOTES |
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1 Supported by grants from NIH (Grant CA09141),
the U.S. Department of Defense Congressionally Funded Breast Cancer
Research Program (Grant BC980871), and a career development award from
the Society for Surgery of the Alimentary Tract (all to J. A. D.). H. R. is supported by a postdoctoral training award from the
Susan Komen Foundation. 
2 To whom requests for reprints should be
addressed, at Department of Surgery, Box 8109, Washington University
School of Medicine, St. Louis, MO 63110. E-mail: drebinj{at}msnotes.wustl.edu
3 The abbreviations used are: ODN,
oligodeoxynucleotide; PARP, poly (ADP ribose) polymerase.
4 Roh et al., unpublished
results.
Received 9/30/99. Accepted 12/14/99.
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REFERENCES |
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), or scrambled antisense (
) ODNs in various doses in
triplicate. Cells were counted 5 days after treatment. Results show
means; bars, SE. C, sequence-specific effects
of HER2/neu antisenseODNs on p185 expression-Western blot
analysis. Two µg each of protein lysates from cells treated with
Lipofectin with or without antisense (HAS), sense
(HS), scrambled antisense (HSC), one-base
mismatched antisense (1 MM), or four-base mismatched
antisense (4 MM) ODNs were separated on a 7.5% SDS-PAGE
and transferred onto PVDF membrane. The HER2/neu encoded
p185 molecule was identified by probing with an
anti-HER2/neu monoclonal antibody. The same blot was
reprobed with an antiactin antibody to control for protein loading.
D, sequence-specific effects of HER2/neu
antisense ODNs on cell growth. Cells were treated in triplicate as
described above and counted 5 days after treatment. Results show means;
bars, SE.
