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Int. J. Cancer: 72, 631–636 (1997)
r 1997 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
Publication de l’Union Internationale Contre le Cancer
INHIBITION OF TUMORIGENICITY IN LUNG ADENOCARCINOMA
CELLS BY c-erbB-2 ANTISENSE EXPRESSION
Patrizia CASALINI1, Sylvie MÉNARD1, Sergio M.I. MALANDRIN1, Cristina M. RIGO1, Maria I. COLNAGHI1*,
Constance M. CULTRARO2 and Shoshana SEGAL2
1Division of Experimental Oncology E, Istituto Nazionale Tumori, Milan, Italy
2NCI-Navy Medical Oncology Branch, Naval Hospital Bethesda, MD, USA
The lung carcinoma cell line Calu3, which overexpresses
the c-erbB-2 oncogene, was stably transfected with antisense
(AS) cDNA constructs encompassing different regions of the
c-erbB-2 gene. Transfected cells were analyzed for their
tumorigenic properties in vitro and in nude mice. Two independent clones, AS F1 (low erbB-2 expressor) and AS B12 (high
erbB-2 expressor), as well as the polyclonal Calu3/AS 58, were
selected for these analyses. In Calu3/AS 58 transfected cells
and in the AS F1 clone, c-erbB-2 RNA and protein levels were
lower than those detected in the parental cell line and the AS
B12 clone. Anchorage-independent growth and tumor take
were also significantly reduced. Furthermore, cells derived
from primary tumors of Calu3/AS 58, AS F1 and AS B12 lost
the AS c-erbB-2 DNA insert but retained the gene for G418
resistance. Our results suggest that a correlation between
c-erbB-2 overexpression and tumorigenicity may exist in the
Calu3 lung carcinoma cell line. Int. J. Cancer 72:631–636, 1997.
r 1997 Wiley-Liss, Inc.
The c-erbB-2/HER-2 proto-oncogene encodes a 185-kDa transmembrane tyrosine kinase receptor (p185c-erbB-2) which is the
human counterpart of the rodent c-neu. Abnormalities of p185c-erbB-2
expression which result primarily from transcriptional deregulation
and/or gene amplification (Pasleau et al., 1993; Kraus et al., 1987)
have been implicated in the pathogenesis of a number of human
tumors, including breast, stomach, ovary, bladder and salivary
gland carcinomas (Hynes and Stern, 1994).
Several retrospective clinical studies of breast adenocarcinoma
patients have indicated that p185c-erbB-2 overexpression correlates
with reduced overall and disease-free survival (Press et al., 1993;
Byers et al., 1990) and a limited response to some adjuvant
cytotoxic and hormonal therapies (Gusterson et al., 1992; Wright et
al., 1989). A role for p185c-erbB-2 in neoplastic transformation of
NIH3T3 cells (Di Fiore et al., 1987) and in the induction of a
tumorigenic phenotype in the immortalized MTSV1.7 human
mammary epithelial cell line (D’Souza and Papa-Dimitriou, 1994)
has also been demonstrated. Decreased expression of p185c-erbB-2,
through the use of monoclonal antibodies (MAbs) specific for the
extracellular domain of the protein, resulted in growth inhibition
and, in some cases, differentiation of breast cancer cell lines (Bacus
et al., 1992; Tagliabue et al., 1991; Stancovski et al., 1991). In
addition, studies have shown that c-erbB-2 interaction with the
adenovirus E1A promoter suppresses tumorigenicity (Yu et al., 1990),
although no evidence was found to indicate that the loss of tumorigenicity was the direct consequence of this interaction (Yu et al., 1993).
Antisense (AS) RNA has been shown to be an effective tool to
directly control the expression of target gene products without
affecting other cellular functions (Hélène, 1994). AS oligonucleotides have been used to inhibit the in vitro proliferation of human
breast cancer cells in which c-erbB-2 is amplified (Colomer et al.,
1994), but it remains unclear whether such inhibition is reflected in
decreased tumorigenicity in vivo. Moreover, specific downregulation of c-erbB-2 mRNA and protein has been achieved using
phosphorothioate AS oligonucleotides, resulting in accumulation
of cells in the G1 phase of the cell cycle (Vaughn et al., 1995).
To test whether inhibition of c-erbB-2 expression correlates with
reduced tumorigenicity in vivo, the human Calu3 lung carcinoma
cell line was transfected with AS cDNA constructs encompassing
various regions of the c-erbB-2 gene. Growth properties of the
transfectants as well as their ability to induce tumors in nude mice
were examined.
MATERIAL AND METHODS
Preparation of constructs
The LTR-2/erbB-2 expression vector, kindly provided by Dr. P.P.
Di Fiore (Di Fiore et al., 1987), was used as a template to obtain the
following fragments: 1) 151-bp PCR product corresponding to the
58 end (nts 1–151) of the human c-erbB-2 coding region; 2) 291-bp
PCR product corresponding to the transmembrane region (TM) (nts
2110–2400) of the human c-erbB-2 coding region; and 3) 415-bp
PCR product corresponding to the 38 end (nts 3241–3655) of the
human c-erbB-2 coding region. cDNA fragments were amplified
using synthetic primers (Fig. 1a) containing HindIII restriction
sites (Fig. 1a, boldface). Amplifications were performed in a 100-µl
volume containing 0.3 mM of each primer and 2.5 units of Taq
polymerase (Perkin Elmer Cetus, Norwalk, CT); the amplification
program used was 1 min at 94°C, 1 min at 65°C and 2 min at 72°C
for 30 cycles followed by 10 min at 72°C. The amplified cDNA
fragments were ligated into HindIII-digested pRC/CMV vector
(Invitrogen, San Diego, CA), and one clone of each construct in the
antisense orientation (pRC/CMV-AS 58, pRC/CMV-AS TM and
pRC/CMV-AS 38) was selected. Sequence analysis of the 3 selected
clones showed 100% homology with the published sequence of
c-erbB-2 (Coussens et al., 1985) (GenBank A.N. M11730).
Transfection and selection
The human lung adenocarcinoma cell line Calu3 (ATCC,
Rockville, MD) with the amplified and overexpressed c-erbB-2
gene (Ménard et al., 1993) was used. The cell line was maintained
in RPMI-1640 (Sigma, St. Louis, MO) medium supplemented with
10% FCS (HyClone, Logan, UT) and glutamine at 37°C in 5% CO2
incubator. Cells were plated in 100-mm dishes, and when 80%
confluent, transfected using the Lipofectin (GIBCO BRL, Paisley,
UK) procedure; cells were treated either with 50 µl of Lipofectin
reagent alone for selection control, or with Lipofectin plus 20 µg of
pRC/CMV or pRC/CMV-AS 58 in serum-free medium. The
resulting transfectants were designated as Calu3/neo and Calu3/AS
58, respectively. Cells were then incubated for 16–18 hr at 37°C
and, after fresh medium replacement, were incubated for an
additional 24 hr. To select for clones, cells were trypsinized and
plated in the presence of 500 µg/ml of G418 (GIBCO BRL). When
all control cells died, medium was replaced and transfectants
maintained in medium supplemented with 300 µg/ml of G418.
Surviving resistant colonies were harvested, grown and pooled. An
aliquot of the Calu3/AS 58 pool was used for single-cell cloning by
limiting dilution.
Contract grant sponsor: Associazone Italiana per la Ricerca sul Cancro;
Contract grant sponsor: CNR-ACRO.
*Correspondence to: Division of Experimental Oncology E, Istituto
Nazionale Tumori, Via Venezian 1, 20133 Milan, Italy. Fax: 139-22362692. E-mail: [email protected]
Received 19 December 1996; revised 18 March 1997
632
CASALINI ET AL.
FIGURE 1 – Construction and integration analysis strategies. (a) Schematic representation of the construction of pRC/CMV-AS 58,
pRC/CMV-AS TM and pRC/CMV-AS 38 expression vectors. The AS fragments were amplified from the LTR-2/erbB-2 expression vector using
synthetic primers (HindIII recognition sequence in bold). (b) Schematic of primer annealing sites for construct integration analysis by PCR and
antisense mRNA detection by RT-PCR.
Construct integration analysis
Genomic DNA was extracted from cultured cells using standard
procedures (Ausubel et al., 1987). For construct integration
analysis, a 180-bp fragment was amplified by PCR (see above)
using a vector-specific T7 oligonucleotide as an upstream primer
and a c-erbB-2-specific oligonucleotide as a downstream primer
(Fig. 1b). Genomic DNAs (0.5 µg) obtained from parental or
transfected cells or 50 ng of control plasmid were used as
templates. The amplification program used was an initial denaturation step for 5 min at 95°C, followed by 1 min at 94°C, 1 min at
50°C and 2 min at 72°C for 30 cycles and a final extension step for
10 min at 72°C. PCR products (30 µl) were fractionated on a 1.5%
INHIBITION OF TUMORIGENICITY BY c-erbB-2 ANTISENSE
agarose gel, transferred to a nitrocellulose membrane (Schleicher
and Schuell, Keene, NH) and hybridized to a [32P]dCTP (Amersham, Aylesbury, UK) random-primed (Boehringer Mannheim,
Mannheim, Germany) c-erbB-2 probe encompassing nt 1–151.
Reverse transcription and RT-PCR
Total RNA (0.1 µg) (see Northern Blot Analysis) was reversetranscribed at 50°C for 15 min in a volume of 20 µl using 50 U of
M-MLV reverse transcriptase (Perkin Elmer Cetus), 1 mM of each
dNTP and 4 mM of SP6 primer (vector-specific) (Fig. 1b). The
enzyme was then heat-inactivated and cDNAs were amplified
using T7 and the specific c-erbB-2 primers as described in the
procedure for construct integration analysis (except that annealing
was done at 50°C). Amplified material (30 µl) was fractionated on a
1.5% agarose gel, and Southern blot analysis was performed as
described above.
Northern blot analysis
Total RNA was purified from cells using the Ultraspec RNA
isolation system (BioTecx, Houston, TX). Aliquots of 30 µg were
fractionated on a 1% agarose-formaldehyde gel, transferred to
nitrocellulose membranes (Schleicher and Schuell), and hybridized
to a [32P]-labeled-c-erbB-2 coding region probe. After stripping,
membranes were rehybridized to a [32P]-labeled-b-actin probe
(Amersham) (Gunning et al., 1983). Densitometric analysis of
membranes was performed by scanning on a phosphorimager using
the Image Quant System (Molecular Dynamics, Sunnyvale, CA).
Flow cytometric analysis
Cells were trypsinized, washed with fluorescence-activated cell
sorting (FACS) buffer (PBS containing 0.03% of BSA) and
resuspended in 100 µl FACS buffer containing 10 µg/ml of
anti-p185c-erbB-2 MAb MGR6 (Centis et al., 1992). Following
incubation on ice for 45 min, cells were washed with FACS buffer
and incubated with fluorescein-labeled goat anti-mouse Ig (Kierkegard and Perry, Gaithersburg, MD) for 45 min on ice. After washing
with FACS buffer, cells were resuspended in 1 ml FACS buffer and
analyzed for fluorescence using a Becton Dickinson FACScan
(Mountain View, CA). Fluorescence intensity for the c-erbB-2 oncoprotein was calibrated using quantitative radio-immunoassay of different
cell lines with known protein expression (Pupa et al., 1993).
633
RESULTS
Effects of different c-erbB-2 antisense constructs
on cell proliferation
Three constructs, one consisting of 151 bp derived from the 58
end (AS 58-pRC/CMV), another of 300 bp corresponding to the
transmembrane region (AS TM-pRC/CMV) and the third of 450 bp
encompassing the 38 region (AS 38-pRC/CMV) of the c-erbB-2
c-DNA, were cloned independently in the antisense orientation into
the pRC/CMV expression vector (Fig. 1). To test the effect of
c-erbB-2 AS expression on cell proliferation, Calu3 cells, which
constitutively overexpress c-erbB-2, were transfected with the 3
recombinant plasmids and with an empty pRC/CMV vector as a
control, and colony formation in each of the transfected cell
populations was evaluated. Two days after transfection, cells were
plated at 3.5 3 105 (cells/plate) and grown in the presence of G418
for 3–4 weeks. Colonies consisting of at least 10 cells were then
counted. The colony-forming ability of Calu3 cells transfected with
each of the 3 AS vectors was markedly reduced as compared to
cells transfected with the empty vector, but the largest reduction
observed was in cells transfected with the AS 58-pRC/CMV
construct (Fig. 2), as repeatedly confirmed in other experiments
(data not shown). Thus, all remaining experiments were performed
using this construct.
Construct integration and its effect
on c-erbB-2 expression in Calu3 cells
Colonies of Calu3 cells stably transfected with the AS 58-pRC/
CMV vector (Calu3/AS 58) were pooled and grown for further
studies. This Calu3/AS 58 polyclonal cell line was also subcloned
by limiting dilution. Analysis of 10 independent clones obtained by
this procedure revealed a wide range of c-erbB-2 expression at both
the mRNA and protein levels. Two clones, AS F1 (c-erbB-2 lowest
expressor) and AS B12 (c-erbB-2 highest expressor), together with
the original polyclonal cell line Calu3/AS 58 and the empty
vector-transfected Calu3 (Calu3/neo), were used for further analyses. Construct integration was examined by PCR analysis of
Soft agar assay
Cells were plated in triplicate wells of 6-well plates (Costar,
Celbio, Milan, Italy) in semi-solid medium containing 0.3%
Bacto-Agar (DIFCO, Detroit, MI) supplemented with 30% FCS.
Medium for transfectants was supplemented with 300 µg/ml G418.
Plates were incubated at 37°C in 5% CO2 and colonies were scored after
3 weeks. Statistical analysis was performed using a two-tailed t-test.
Tumor growth assay
The tumorigenic capacity of transfected and parental Calu3 cells
was assayed in 2 series of experiments following s.c. injection of
1 3 105 cells into the flanks of nude mice. In the first series,
parental Calu3, AS F1 and AS B12 cells were compared, and in the
second series Calu3, Calu3/neo and AS 58 cells were compared.
Tumor volume was calculated using the formula 0.5 3 d21 3 d2,
where d1 is the smaller and d2 the larger diameter of the tumor as
measured with calipers. Animals were killed when the average
tumor size reached 1 cm3, or earlier if the tumor became necrotic.
Statistical analysis on tumor take results was performed for every
series using the chi-square test. After sacrificing the mice, one of
each randomly selected primary tumors was re-established in
culture and then tested for G418 resistance and c-erbB-2 expression. The remaining tumors were snap-frozen by immersion in
liquid nitrogen immediately following removal and stored at
280°C prior to RNA isolation. RNAs were isolated from tumor
tissue using the Ultraspec isolation system (BioTecx).
FIGURE 2 – Colony-forming potential of Calu3 cells transfected with
pRC/CMV-AS 58, pRC/CMV-AS TM or pRC/CMV-AS 38. After
transfection, cells were plated at 3 3 105 cells/well in the presence of
300 µg/ml G418. After 3 weeks, colonies were stained with methylene
blue. Data are reported as percentage of colonies obtained in control
cells transfected with the empty vector. Error bars represent standard
deviation of 3–6 replicates.
634
CASALINI ET AL.
genomic DNA using a 58 T7 vector-specific oligonucleotide and a
38 c-erbB-2 oligonucleotide spanning nucleotides 115–141 (Fig.
1b). Southern analysis with a c-erbB-2 cDNA probe supported the
specificity of the PCR product obtained (Fig. 3). The intensity of
the hybridization signal indicated that the level of construct
integration in the 2 AS clones was similar to that of the polyclonal
Calu3/AS 58. The set of primers specifically amplified transfected
AS c-erbB-2 and, as expected, no PCR product was generated with
parental Calu3 genomic DNA.
To determine whether this AS inhibits c-erbB-2 expression, total
RNA obtained from polyclonal Calu3/neo, Calu3/AS 58 and clones
AS F1 and AS B12 was analyzed by Northern blotting, with
densitometry normalized to actin levels (Fig. 4). The polyclonal
Calu3/AS 58 showed only a slightly lower level of c-erbB-2 mRNA
than parental Calu3 or control Calu3/neo cells. The AS F1 clone
demonstrated a 2.2-fold reduction as compared to the Calu3/neo
cells, whereas c-erbB-2 expression in the AS B12 clone was
unaffected. AS c-erbB-2 mRNA was undetectable by Northern blot
analysis (data not shown). Because antisense transcripts may not
accumulate to levels detected by Northern blot analysis due to rapid
degradation, we utilized the more sensitive RT-PCR assay followed
by Southern blot analysis. As shown in Figure 5, AS mRNA
expression was lower in the AS B12 clone than in the AS F1 clone.
FACScan analyses to determine whether the reduction in
c-erbB-2 mRNA was paralleled by a reduction in p185c-erbB-2
membrane protein expression (Fig. 6) revealed a wide peak for the
Calu3/AS 58 polyclonal cells, indicating a high degree of heterogeneity in p185c-erbB-2 expression, consistent with the similarity of a
majority of cells to the Calu3/neo and parental cells, and with a
small fraction of cells exhibiting reduced oncoprotein expression.
Based on the linear relationship between fluorescence intensity and
amount of oncoprotein (Pupa et al., 1993), we estimated a 70%
decrease in c-erbB-2 expression in this fraction. In contrast, a
homogeneous peak was obtained for the AS F1 cells, with a lower
level of p185c-erbB-2 expression as compared with those in AS B12
cells. The mean intensity decreased from 300 in the control to 100
in F1 cells, corresponding to a 67% reduction. The protein:mRNA
ratio was therefore lower in the F1 clone (0.6) than in the B12 clone
(1) and the parental cells (1).
Reduction in tumor development by antisense c-erbB-2
The tumorigenic capacity of the AS clones and polyclonal
Calu3/AS 58 was evaluated by assaying anchorage-independent
growth and/or subcutaneous growth in nude mice (Table I). Both
the clonogenic potential and the tumor take of the AS F1 cells were
significantly lower than the values obtained for parental Calu3 or
FIGURE 3 – Southern blot analysis of PCR products derived from
genomic DNAs. PCR products, obtained as described in Figure 1b,
were fractionated on a 1.5% agarose gel, transferred to nitrocellulose
filters and hybridized with a [32P]-dCTP-labeled c-erbB-2 probe
encompassing nucleotides 1–151. Lane 1, parental Calu3 cell line; lane
2, clone AS B12; lane 3, clone AS F1; lane 4, polyclonal Calu3/AS 58;
lane 5, pRC/CMV-AS 58 control plasmid.
AS B12 cells. Indeed, the AS F1 cells exhibited a 3-fold reduction
in anchorage-independent growth as compared to AS B12 and
Calu3 cells. After subcutaneous injection of 105 AS F1 cells or
Calu3/AS 58 cells, only 6 of 13 and 9 of 15 nude mice developed
measurable nodules, respectively. Nude mice injected with AS B12
or Calu3/neo control cells displayed 85% and 100% tumor take,
respectively, whereas parental Calu3 cells induced tumors in
93–100% of the mice. The growth properties of tumors developing
from AS F1 or AS B12 cells were very similar to those of the
parental Calu3 tumors, except that measurable tumors from AS F1
arose 10 days later than those from parental cells. Northern blot
analysis of tumor RNAs derived from antisense transfectants after
in vivo growth showed that endogenous levels of c-erbB-2
expression were identical to those in controls (data not shown).
RT-PCR analysis clearly demonstrated that tumor cells derived
from primary tumors of Calu3/AS 58, AS F1 and AS B12 lost the
AS c-erbB-2 DNA insert but retained the gene for G418 resistance
(Fig. 7).
DISCUSSION
The present study was designed to test whether the c-erbB-2
oncogene plays a role in determining the transformed phenotype of
FIGURE 4 – Northern blot analysis of parental and transfected Calu3
cells. Total RNA (30 µg) was fractionated on a 1% agaroseformaldehyde gel, transferred to a nitrocellulose filter and hybridized
with [32P]dCTP-full-length erbB-2 and a [32P]dCTP-labelled b-actin
control. Histograms show results of densitometric analysis expressed in
arbitrary units and normalized to b-actin expression. Lane 1, parental
Calu3 cell line; lane 2, clone AS B12; lane 3, clone AS F1; lane 4,
polyclonal Calu3/AS 58; lane 5, Calu3/neo.
FIGURE 5 – Southern blot analysis of RT-PCR products derived from
total RNA. Total RNAs (1 µg) were reverse-transcribed and amplified
by PCR as described in Figure 1b. The amplified products were
fractionated on a 1.5% agarose gel, transferred to a nitrocellulose filter
and hybridized with the same [32P]-dCTP-labeled c-erbB-2 probe used
for Figure 3. Lane 1, Calu3/neo cell line; lane 2, polyclonal Calu3/AS
58; lane 3, clone AS F1; lane 4, clone AS B12.
INHIBITION OF TUMORIGENICITY BY c-erbB-2 ANTISENSE
635
FIGURE 6 – FACScan analysis of parental and transfected Calu3 cells. (a) (1) parental Calu3, (2) Calu3/neo, (3) Calu3/AS 58. (b) (1) parental
Calu3, (4) AS F1, (5) AS B12.
TABLE I – CLONOGENICITY AND TUMOR TAKE OF TRANSFECTED CELLS1
Target cells
Clonogenicity
Calu3
AS B12
AS F1
Calu3
Calu3/neo
Calu3/AS 58
112 6 31.82
140 6 16.97
38 6 4.24
N.D.
N.D.
N.D.
p2
0.442
0.016
Tumor take
%
13/14
11/13
6/13
14/14
9/9
9/15
93
85
46
100
100
60
p3
0.01
0.005
1In
clonogenicity experiments, cells were plated in triplicate in 0.3%
Bacto-Agar and scored after 3 weeks. In tumorigenicity experiments,
105 cells were injected subcutaneously in the flank of 9–15 nude mice.
Tumor take was scored when control tumors reached 1,000 mm3.–
2Evaluated by two-tailed t-test.–3Evaluated by chi-square test.
Calu3 cells. The results suggest that a correlation between c-erbB-2
overexpression and tumorigenicity may exist in these cells. Reduced expression of the oncoprotein, due to expression of AS
c-erbB-2, resulted in reduced clonogenicity and reduced tumor take
in vivo. Inhibition of c-erbB-2 expression by the AS construct
indicates that c-erbB-2 overexpression is an important factor
required for the growth of this human lung adenocarcinoma cell
line. This requirement was evidenced by the reduced ability of AS
transfectants to form colonies. Therefore, it appears that inhibition
of c-erbB-2 overexpression is detrimental to cells that normally
overexpress the gene. Our findings are consistent with recent
reports demonstrating that inhibition of c-erbB-2 expression by the
introduction of c-erbB-2-specific antibody into cells leads to
apoptosis (Deshane et al., 1996). It is thus likely that an intermediate expression of antisense constructs in the polyclonal cells and
those derived from the selected clones is able to reduce but not
abolish oncoprotein expression allowing cell survival and growth.
In fact, the growth of these transfectants in soft agar and in nude
mice was reduced but not completely inhibited. The possibility that
the decrease in malignancy observed in the AS F1 clone was due to
the selection of a pre-existing poorly malignant cell present in the
parental Calu3 cannot be completely ruled out; however, random
selection of cells with low proliferation potential from rapidly
growing cells seems unlikely. Moreover, all clones isolated from
the parental Calu3 cells by limiting dilution overexpressed
FIGURE 7 – Southern blot analysis of RT-PCR products derived from
total RNA. Total RNA (1 µg) from transfected cells and tumors was
processed as described in Figure 5. Lane 1, Calu3/neo control cell line;
lane 2, polyclonal Calu3/AS 58 line; lane 3, clone AS F1; lane 4, clone
AS B12; lanes 5–8 correspond, respectively, to lanes 1–4, except for
using RNA extracted from tumors derived from the transfected cell
lines.
c-erbB-2 at levels similar to those observed in the parental cells.
The various patterns of behavior observed in the polyclonal
transfected population further support the relevance of the antisense transfection in reducing c-erbB-2 expression.
The role c-erbB-2 plays in tumorigenicity was confirmed by
RT-PCR of tumor mRNAs, since only the cells that lost the AS
insert were able to establish and develop tumors (Fig. 7). In fact,
the delay in tumor development reflects a lag in time due to the
selection of cells that lost the AS insert. Nevertheless, these cells
remained neomycin-resistant. Immunohistochemical analysis of
the frozen specimens for c-erbB-2 expression revealed homogeneous and comparable staining of the tumors despite the origin of
the cells or the size of the nodule (data not shown).
Ideally, the targeted mRNA and protein are most effectively
inhibited in clones with the highest level of construct integration
and antisense RNA expression. Indeed, AS F1 and B12 cells
display comparable levels of construct integration, but inhibition of
c-erbB-2 expression was observed only in clone F1, which
expresses high levels of AS RNA. Neither the inability to detect AS
RNA by Northern blot analysis nor the loose correlation between
636
CASALINI ET AL.
the level of AS RNA expression/construct integration and the
degree of reduction in c-erbB-2 mRNA was totally unexpected,
since these discrepancies have been observed in other models
(Kook et al., 1994; Neckers et al., 1992; Kasid et al., 1989; van der
Krol et al., 1988). Possible explanations for these findings include
positional effects that may influence the level of transgene transcription, high instability of the RNA duplexes and a greater instability
of the antisense RNA relative to the mRNA such that antisense
RNA is less easily detectable (Kook et al., 1994). In fact, we were
unable to detect AS RNA by Northern analysis even with polyA
RNA, or by RT-PCR using polyT oligonucleotides. Furthermore,
RT-PCR of cytoplasmic RNA with the same oligonucleotide
primers used in RT-PCR of total RNA was also negative, suggesting that AS c-erbB-2 functions in part via transcriptional inhibition
by the formation of DNA/RNA duplexes and/or by inhibition of
mRNA transport to the cytoplasm, forming RNA/RNA hybrids.
Our results, together with similar reports on c-raf-1, EGFR and
uPAR antisense inhibition (Kook et al., 1994; Moroni et al., 1992;
Kasid et al., 1989), suggest the potential utilization of AS approaches for
therapeutic purposes to downmodulate expression of tumor-promoting
genes. However, technical problems such as antisense instability and
low transfection efficiency must first be solved.
ACKNOWLEDGEMENTS
We thank Mrs. D. Labadini for preparing the manuscript and Mr.
M. Azzini for photographic reproduction. This work was partially
supported by a grant from the Associazione Italiana per la Ricerca
sul Cancro and by CNR-ACRO.
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