From Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center-108, Houston, Texas 77030
Received for publication, September 18, 2000, and in revised form, December 14, 2000
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ABSTRACT |
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Epidermal growth factor (EGF) family of growth
factors and their receptors regulate normal and cancerous epithelial
cell proliferation, a process that can be suppressed by
antireceptor blocking antibodies. To identify genes whose
expression may be modulated by antireceptor blocking antibodies, we
performed a differential display screen with cells grown in the
presence or absence of antireceptor blocking antibodies; isolates from
one cDNA clone were 100% identical to human heterogeneous nuclear
ribonucleoprotein K (hnRNP K), a protein with a conserved KH motif and
RGG boxes, has been implicated in such functions as sequence-specific
DNA binding, transcription, RNA binding, and nucleocytoplasmic
shuttling. Both EGF and heregulin- Growth factors and their receptors play an important role in
regulating proliferation of epithelial cells. Abnormalities in the
expression, structure, or activity of their proto-oncogene products
contribute to the development and pathogenesis of cancer. For example,
the human epidermal growth factor
receptor, HER1,1
is overexpressed in a large number of epithelial tumor cells (1). HER2,
the second member of the HER family, shares extensive sequence homology
with the tyrosine kinase domain of HER1 (1-3) and is overexpressed,
amplified, or both in a number of human malignancies, including breast.
Recently, HER3 and HER4 have been added to this receptor family (2, 3).
Regulation of these receptor family members is complex, and they can be
trans-activated in a ligand-dependent manner. For example,
binding of heregulin- Since growth factors regulate the proliferation of cancer cells by
activating cell-surface receptors, one approach to controlling cell
proliferation is to use antireceptor-blocking monoclonal antibodies to
interfere with growth factor receptor-mediated autocrine or paracrine
growth stimulation (6). C225, the human murine chimeric antibody
against the EGF receptor (EGFR), blocks binding of ligand and prevents
ligand-induced activation of receptor tyrosine kinase (6, 7). C225
therapy has been success in phase I and phase IIA multicenter clinical
trials in combination with chemotherapy or radiation (8-11).
Similarly, the humanized form of anti-HER2 recombinant monoclonal
antibody (HCT) inhibits the growth of breast cancer cells
overexpressing HER2 (12) and is currently being used as an effective
drug against breast cancer both alone (13) and in combination with
chemotherapy (14). Antireceptor antibodies inhibit many processes,
including mitogenesis, cell-cycle progression, invasion and metastasis,
angiogenesis, and DNA repair (8).
In eukaryotic cells, heterogeneous nuclear RNAs (hnRNAs), from which
mRNAs are generated by RNA processing, associate with specific
nuclear proteins to form large hnRNP complexes (15, 16). These hnRNP
proteins bind pre-mRNAs and are believed to play important roles in
mRNA biogenesis (17, 18), nucleocytoplasmic transport of mRNA
(19-21), and cytoplasmic mRNA trafficking (22). To date, about 20 hnRNPs (A through U) have been identified. HnRNP K, the major
poly(iC)-binding protein, has several structural features not shared
with other hnRNP proteins (23). For example, the binding of hnRNP K to
nucleic acid is mediated by three repeat motifs termed the KH (K
homology) domains rather than by the consensus RNA-binding sequence
found in other hnRNP proteins (23, 24). The KH domain is an
evolutionarily conserved RNA-binding motif also found in fragile X
protein FMR1 (25), meiosis-specific splicing factor MER1 (26), and
paraneoplastic Ri autoantigen (27). In addition, hnRNP K binds
single-stranded DNA in vitro (28) and has been identified as
a sequence-specific DNA-binding protein (29), consistent with its
proposed role in transcription. More recently, hnRNP K has been shown
to bind to a cis-element in the human c-myc promoter (30)
and to activate c-myc expression (31) by promoting the
synthesis of c-myc mRNA from a reporter gene (32). The
hnRNP K protein can also interact with some proto-oncogene products and
to act as a docking platform in signal transduction cascades (33, 34).
The potential involvement of hnRNP in transformation was suspected,
four splice variants of hnRNP K are up-regulated in SV40-transformed
cells (35), and hnRNP B1 is the only other member of the hnRNP family,
beside hnRNP K that is overexpressed in human lung cancers (36).
Despite the widely believed involvement of hnRNP K in
post-transcriptional control, its possible regulation by the EGF family
and by therapeutic antireceptor antibodies remains unexplored.
To identify genes whose expression may be modulated by the activity of
the EGF family of receptors because of ligand-induced activation
of receptor tyrosine kinases or inhibition of receptor-associated tyrosine kinase activation, we used a human cDNA array approach to
isolate cDNAs differentially expressed in the presence and absence
of antireceptor antibodies. We identified one clone that was identical
to human hnRNP K. In human breast cancer cells, EGF and HRG induced
hnRNP K mRNA and protein expression that could be effectively
blocked by pretreatment with antireceptor monoclonal antibodies. Our
results also suggested that hnRNP K is involved in EGF- or HRG-induced
transcription from the c-myc promoter and that hnRNP K
expression can be used as a molecular monitor to assess the anti-tumor
action of therapeutic antireceptor agents.
Cell Cultures and Reagents--
Human colon cancer DiFi and FET
cells, and breast cancer MCF-7, MDA-468, BT-474, SK-BR3, MDA-231, and
MDA-435 cells and vulvar carcinoma A431 cells, were maintained in
Dulbecco's modified Eagle's medium-F-12 (1:1) supplemented with 10%
fetal calf serum. C225 and HCT ware provided by Imclone Systems,
Inc. and Genentech Inc., respectively. Recombinant HRG- Gene Discovery Array Screening--
DiFi and FET cells were
cultured with or without C225 for 10 h, and total RNA was isolated
using TRIZOL reagent (Life Technologies, Inc.). Poly(A) RNA was
isolated using a poly(A) RNA isolation kit (Invitrogen). Gene discovery
array (GDA) filters (version 1.2) containing 18,376 nonredundant human
cDNA clones were purchased from Genome Systems. Probe preparation
and hybridization conditions were performed according to
manufacturer's recommendations. In brief, 2 µg of poly(A) RNA was
reverse-transcribed using T18MN primer (Genome Systems) and Superscript
II reverse transcriptase (Life Technologies, Inc.) in the presence of
[33P]dCTP. cDNA was purified using a G-50 spin
column, treated with 0.25 M NaOH to remove RNA, and
neutralized with Tris. Identical filters were hybridized to cDNA
probes in 50% formamide buffer at 42 °C for 16 h, washed two
times with 2× SSC, and washed two more with 0.6× SSC. Filters were
exposed to a phosphoimager, and images were quantitated by the
bioinformatics department at Genome Systems using Array Vision
software. Normalized intensity values for control and C225-treated
filters were compared with determine -fold induction.
Cell Extracts, Immunoblotting, and Immunoprecipitation--
To
prepare cell extracts, cells were washed three times with
phosphate-buffered saline and lysed in buffer (50 mM
Tris-HCl, pH 7.5, 120 mM NaCl, 0.5% Nonidet P-40, 100 mM NaF, 200 mM NaVO5, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin) for 15 min on ice. The lysates were centrifuged in an Eppendorf centrifuge at
4 °C for 15 min. Cell lysates containing equal amounts of protein were resolved by SDS-PAGE, transferred to nitrocellulose, and probed
with the appropriate antibodies. Equal numbers of cells were
metabolically labeled for 4-8 h with 100 µCi/ml
[35S]methionine in methionine-free medium containing 2%
dialyzed fetal bovine serum in the absence or presence of indicated
treatments. Cell extracts containing equal trichloroacetic acid
perceptible counts were resolved on SDS-PAGE, immunoprecipitated with
the desired antibody, and analyzed after exposure to film.
Northern Hybridization--
Total cytoplasmic RNA was isolated
using the TRIZOL reagent, and 20 µg of RNA was analyzed by
Northern hybridization using a cDNA fragment of human hnRNP K. Ribosomal RNA (28 and 18 S) was used to assess the integrity of the RNA
and for RNA-loading controls; blots were also routinely reprobed with
control glyceraldehyde-3-phosphate dehydrogenase cDNA.
Human Tissue Samples--
Human breast tissue samples were
obtained from a tissue bank maintained by Dr. Luis Costa (Hospital de
Santa Maria, Lisbon, Portugal). Specimens derived from patients who had
undergone surgery for breast cancer were frozen in liquid nitrogen and
stored at Xenografts Studies--
A431 cells (107) were
implanted subcutaneously into nude mice and allowed to grow for 8 days.
The mice were given intraperitoneal injections of either
phosphate-buffered saline or C225, 1 mg/mouse, twice a week for 3 weeks. Treatment of C225 alone resulted in transient inhibition of
tumor growth. Tumor size was measured twice weekly with calipers, using
the formula Identification of hnRNP K as an Antireceptor Antibody-regulated
Gene--
In an attempt to identify genes whose expression may be
modulated in human cancer cells by antireceptor antibodies that block receptor activity, we used GDA filters human version 1.2 obtained from
Genome Systems. GDA is a single nylon filter spotted with 18,376 nonredundant human cDNA clones chosen from the Integrated Molecular
Analysis of Genome and Expression collection. We have initially
used two cell lines: high EGFR overexpressing DiFi cell line and a
moderate EGFR expressing FET cell line (38). Cells were cultured with
or without anti-EGF receptor antibody C225 for 10 h, total RNA was
isolated, and cDNA probes were prepared using reverse
transcription. The filters were hybridized and exposed to a
phosphoimager. Phosphoimager scans were sent to Genome Systems, and
their Bioinformatics Department used Array Vision software to analyze
differential expression of genes. This analysis identified several
differentially expressed gene products. Representative data for 11 genes whose expression was altered are shown in Fig. 1. To further characterize these
antibody-responsive genes, we obtained expressed sequence tag clones
arrayed on GDA filters from Genome Systems. While using the cDNAs
obtained, we uncovered the problem that the clones received from Genome
Systems did not sequence verify to data base numbers, and we got mixed
results in our Northern blots. Upon contacting Genome Systems, we found that their GDA version 1.2 filters were not sequence-verified, were
developed on the basis of information from Integrated Molecular Analysis of Genome and Expression consortium clones, and were expected to have mixed colony stocks. (GDA1.2 filters are discontinued due to these problems). To resolve this issue, we isolated multiple single colonies from each clone stock and analyzed them by
Northern analysis. Northern analysis of single colony-isolated
cDNAs showed that one of the cDNAs was down-regulated by
antibody treatment. Sequencing of the cDNA revealed that it had
extensive similarity with hnRNP K; however it possessed an extra region
of 400 base pairs, suggesting that this was an isoform of hnRNP K. Since these expressed sequence tag clones were isolated from fetal
liver, we screened a mammary gland cDNA library
(CLONTECH) with the 1.1-kilobase pair
cDNA probe to verify that this isoform of hnRNP K was expressed in
the mammary gland. Ten positive clones were isolated and sequenced. Comparison of the sequences with the GenBankTM revealed
that the sequences were 100% identical to human hnRNP K gene.
To confirm that the hnRNP K gene was regulated by growth factors in
mammary epithelial cells, regulation of the hnRNP K gene expression by
antibodies was verified by experiments involving Northern analysis
using the full-length cDNA (1.8 kilobase pairs) isolated
from mammary gland (hnRNP K, identical GenBankTM to
accession number S74678). A431 cells, which overexpress EGFR, are
growth-stimulated by autocrine transforming growth factor- Regulation of hnRNP K Expression by Growth Factors--
Since
MDA-231 cells are known to constitutively secrete HRG, a ligand for
HER3 and HER4 that can transactivate EGFR and HER2, the above results
raised the possibility of HRG regulation of hnRNP K mRNA
expression. HRG treatment of human breast cancer MCF-7 (38), and
colorectal cancer LS174 and CaCO2 cells (39), was accompanied by
increased expression of hnRNP K mRNA (Fig. 2, A and B). EGF
regulation of hnRNP K mRNA expression was also confirmed using a
mouse NIH3T3 cell line (HER14) that stably expressed human EGFR,
lack production of TGF-
Western blot analysis was performed to determine whether the modulation
of hnRNP K mRNA levels by growth factors and antireceptor monoclonal antibodies was associated with a corresponding modulation in
the expression of hnRNP K protein. Results demonstrated that A431 cells
and human colon carcinoma DiFi cells, which have functional TGF- C225 Therapy Modulates hnRNP K Expression in a Xenograft
Model--
Our results presented above suggest that hnRNP K expression
is positively regulated by growth factors and can be reduced by antireceptor-blocking antibodies. To further understand the
significance of hnRNP K expression in tumor cell growth regulation, we
next examined the effect of C225 therapy on the status of hnRNP K
expression in in vivo setting using the A431 xenograft
model. As expected, C225 treatment of well established A431 cell
xenografts was associated with a reduced regression of tumors (Fig.
4A) and appearance of very
smooth boundaries between tumors and surrounding tissues compared with
control untreated tumors (Fig. 4B). Importantly, significant
reductions in the levels of hnRNP K mRNA (Fig. 4C) and
hnRNP K protein (Fig. 4D) accompanied C225 therapy.
hnRNP K Expression in Human Breast Cancer--
Since these results
show that hnRNP K expression is regulated by growth factors, we
explored whether there is any relationship between the proliferation
state in murine and human tissue and hnRNP K expression. Northern blot
analysis of multiple mice tissues revealed the presence of low levels
of hnRNP K mRNA in all tissues. However, the hnRNP K transcript
levels were significantly higher in tissues such as cerebellum, ovary,
and testis (Fig. 5A).
Interestingly, there was expression of only the faster migrating form
of hnRNP K mRNA in testes (Fig. 5A, lane 14).
We also examined the expression of hnRNP K in a small number of human
breast carcinoma biopsy samples (37). In general, grade III tumor
specimens had a higher level of hnRNP K protein compared with grade II
tumor specimens (Fig. 5B). Additional studies utilizing
large number of clinical samples are needed to confirm these
findings.
Effect of hnRNP K on the Biology of Breast Cancer Cells--
Data
from several recent reports indicate that c-myc, a growth
factor-inducible early gene, is one of the downstream targets of hnRNP
K (32). We verified the potential role of hnRNP K in growth
factor-mediated stimulation of transcription from the c-myc promoter using a luciferase reporter gene containing c-Myc promoter. As
expected, cotransfection of hnRNP K up-regulated the c-Myc promoter
activity (Fig. 6A). Treatment
of cells with antireceptor antibodies resulted in a significant
decreased level of c-myc mRNA (Fig. 6B).
To further delineate the potential contribution of hnRNP K in breast
cancer cells, we next established stable MCF-7 clones expressing
T7-tagged hnRNP K or control vector (Fig.
7A). As expected from the data
in Fig. 6, cells expressing hnRNP K also exhibited an increased level
of c-Myc (Fig. 7A) as well as stimulation of transcription
from the c-myc promoter (Fig. 7B). To examine the potential influence of hnRNP K expression on the growth characteristics of breast epithelial cancer cells, we measured the proliferation rate
and ability of cells to grow in an anchorage-independent manner.
Expression of hnRNP K significantly enhanced the proliferation rate of
MCF7 cells (Fig. 7C). Overexpression of hnRNP K was
accompanied by a significant reproducible enhancement of the ability of
cells to form larger colonies in soft agar as compared with those
formed by vector transfected control cells (Fig. 7D).
Together, these observations suggested that breast cancer cells
expressing hnRNP K have altered growth characteristics.
In summary, we provide new evidence that the hnRNP K is a target of
growth factor in human cancer cells and that hnRNP K positively controls the growth rate of human breast cancer cells. This conclusion is based on the following evidence: 1) up-regulation of hnRNP K
mRNA and protein by both HRG and EGF in cells with low levels of
EGF and HER2 receptors; 2) down-regulation of hnRNP K mRNA and
protein by HCT and C225 in cells with high levels of EGF and HER2 receptors; 3) blockage of ligand-induced stimulation of hnRNP K by
an antireceptor antibody in breast cancer cells with low levels of
receptors; 4) a reduction in the level of hnRNP K in tumor xenografts
treated with C225; 5) deregulation of hnRNP K expression cells leads to
an enhancement of the proliferation and anchorage-independent growth of
breast cancer cells; and finally, 6) human breast tumor specimens from
grade III tumors exhibited an increased level of hnRNP-K protein
compared with the levels in grade II tumors.
The molecular mechanisms by which hnRNP K affects the growth rate of
human cells are not clear at the moment. hnRNP K has been shown to
activate the human c-Myc promoter (32) and increases the level of c-Myc
protein. Enhanced expression of c-Myc could enable this transactivation
factor to stimulate its downstream targets genes, leading to cell cycle
progression. In addition, hnRNP K physically interacts with several
oncogene products, including members of the Src protein tyrosine
kinases (33, 34), and this could also contribute to the mitogenic
response. Our demonstrated growth-promoting function of hnRNP K protein
in human breast epithelial cells also supports the observation of hnRNP
K up-regulation in SV40-transformed cells (35). In contrast to these
findings, there are reports showing a repressing effect of hnRNP on
C/EBP-
In summary, our findings have clearly demonstrated for the first time a
potential role of hnRNP K in the actions of HER growth factors that are
widely deregulated in human cancers and that ligand-dependent hnRNP K expression can be effectively
inhibited with antireceptor blocking antibodies C225 and HCT. In
addition, we also provide new evidence that hnRNP K may contribute to
regulating target genes that lead to enhanced growth rate of cancer cells.
1 induced expression of
hnRNP K mRNA and protein in human breast cancer cells. This growth
factor-mediated hnRNP K expression was effectively blocked by
pretreatment of cultures with humanized anti-EGF receptor (EGFR)
antibody C225, or anti-human epidermal growth
factor receptor-2 (HER2) antibody. Anti-EGFR
monoclonal antibody also caused regression of human tumor xenografts
and reduction in hnRNP K levels in athymic mice. Samples from grade III
human breast cancer contained more hnRNP K protein than samples from
grade II cancer. Finally, overexpression of hnRNP K in breast cancer
cells significantly increased target c-myc promoter
activity and c-Myc protein, hnRNP K protein levels, and enhanced breast cancer cell proliferation and growth in an anchorage-independent manner. These results suggested that the activity of human EGF receptor
family members regulates hnRNP K expression by extracellular growth
promoting signals and that therapeutic humanized antibodies against
EGFR and HER2 can effectively block this function.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1 (HRG) to HER3 or HER4 can activate the HER2
as a result of HER2/HER3 or HER4/HER2 heterodimeric interactions
(2-4). HER1 and HER2 have been shown to induce transformation in
recipient cells (2-4), possibly because of excessive activation of
signal transduction pathways. In contrast, transformation by HER3 or
HER4 requires the presence of HER1 or HER2 (5).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1 was
purchased from Neomarkers, Inc. Anti-vinculin antibody and recombinant
EGF was purchased from Sigma. Polyclonal and monoclonal antibodies
against hnRNP K were generously provided by Drs. Pradip Raychaudhuri
and Gideon Dreyfuss (32, 23).
80 °C, as described previously (37). Thawed
tissue samples were homogenized in Triton X-100 lysis buffer (20 mM HEPES, 150 mM NaCl, 1% Triton X-100, 0.1%
deoxycholate (v/w), 2 mM EDTA, 2 mM EDTA, 2 mM sodium orthovanadate, and protease inhibitor mixture (Roche Molecular Biochemicals), and equal amounts of proteins were
analyzed by Western blotting.
/6x larger diameter × (smaller diameter).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Identification of hnRNP K as an antireceptor
antibody-regulated gene. A, representative
differential display patterns of antireceptor-regulated spots on the
GDA filters. Human colon DiFi and FET cells were treated with C225 (50 nM) for 10 h. Total RNAs were isolated and used for
hybridization with the GDA array filters, as described under
"Experimental Procedures." Results are presented as percent change
by antibody treatment over control (CON) untreated cells.
B, MDA231 cells were treated with or without 30 nM TGF- in the absence or presence of 30 nM
C225 or IgG for 30 min. Cell lysates were immunoprecipitated with an
anti-EGFR mAb 528 and immunoblotted with anti-phosphotyrosine mAb 4G10,
and the position of EGFR is shown by an arrow. Sequentially,
the above blot was reprobed with an anti-EGFR mAb to demonstrate the
levels of EGFR in different tubes. C, tumor cell lines were
treated with or without C225 or HCT or IgG (50 nM)
for 16 h. Total RNA was isolated, and the expression of hnRNP K
mRNA was detected by Northern hybridization. Results are
representative of three experiments. GAPDH,
glyceraldehyde-3-phosphate dehydrogenase.
(TGF-
)
and growth-inhibited by C225 (6, 7). To determine the role of growth
factor receptor activity in the regulation of hnRNP, we asked whether
C225 could down-regulate the steady-state level of hnRNP K mRNA.
Earlier studies from this laboratory have shown the effectiveness of
225 antibody to inhibit TGF-
- or EGF-induced tyrosine
phosphorylation of EGFR in DiFi, FET, A431, MDA468, and mouse NIH3T3
cells stably expressing human EGFR (38-41). Consistent with these
reports, C225 also blocked the ability of EGF to stimulate the tyrosine
phosphorylation of EGFR in MDA231 cells (Fig. 1B). Treatment
of A431 or MDA468 cells with C225, but not control IgG, resulted in a
decrease in hnRNP K mRNA expression (Fig. 1C).
Similarly, HCT treatment of BT-474 or SKBR3 cells, which
overexpress HER2, was associated with reduced expression of hnRNP K
mRNA (Fig. 1C). We have earlier demonstrated the ability
of anti-HER2 antibody to suppress the tyrosine phosphorylation
of HER2 in BT-474 and SKBR3 cells (42).
, and to exogenous EGF by growth stimulation
(40). EGF treatment of HER14 cells for 8 h was accompanied by a
significant increase in hnRNP K mRNA levels (data not shown). Taken
together, these results suggest that EGF, HRG, and antireceptor
monoclonal antibodies, which interfere with EGFR and HER2, modulate
hnRNP K mRNA levels in a number of cell types.
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Fig. 2.
Regulation of hnRNP K mRNA expression by
growth factors. Breast cancer MCF-7 (A), and colon
cancer LS174T and CaCO2 cells (B) were treated with HRG (30 nM) for the indicated times. Total RNA was isolated, and
hnRNP K mRNA levels were detected by Northern blot analysis.
Subsequently, the blot was reprobed with a glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) cDNA probe. Quantitation of
mRNA is shown in the bottom panels. Results are
representative of two experiments.
autocrine loop (38), expressed a lower level of the 65-Da hnRNP K
protein, after C225 treatment (Fig.
3A). Similarly, HCT inhibited hnRNP K protein levels in BT-474 cells (Fig. 3A).
In contrast, treatment of MCF-7 cells with EGF or HRG significantly increased the level of hnRNP K protein (Fig. 3B).
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Fig. 3.
Antireceptor antibodies and growth factors
modulate hnRNP K protein level. A, cells were treated
with C225 or HCT (50 nM) for 16 h
(A) and EGF or HRG (30 nM) for the indicated
times (B). Total lysates were run on SDS-PAGE and
blotted with anti-hnRNP K mAb. Anti-vinculin Ab was used as an internal
control. Quantitation of the ratio of hnRNP K: vinculin is shown in the
bottom panel. Results are representative of three
experiments.
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Fig. 4.
Anti-EGF receptor antibody C225 suppresses
hnRNP K expression in a tumor xenograft model. A,
established A431 cells xenografts (0.6-0.8 cm3) were
treated with C225 (100 µg/animal, 2 times a week) or saline for 21 days. Tumor volumes in the control and C225-treated groups at day 29 are shown. B, histology of tumors as assessed by H&E
staining. C, total RNA was isolated from four control and
five antibody-treated tumors. The expression of hnRNP K was immediately
analyzed by Northern hybridization. GAPDH,
glyceraldehyde-3-phosphate dehydrogenase. D, tissue lysates
from four control (C1-C4) and six antibody-treated
(T1-T6) tumors were immediately immunoblotted with an
anti-hnRNP mAb. The blots were reprobed with a control vinculin
antibody.
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Fig. 5.
Expression of hnRNP K in multiple organs and
human breast tumors. A, expression profile of hnRNP K
mRNA in multiple mouse organs as determined by the Northern
hybridization. Ribosomal 28 S and 18 S RNAs were used to assess
the integrity of RNA samples. Lanes: 1, muscle;
2, liver; 3, heart; 4, thymus;
5, colon; 6, kidney; 7, cerebral
cortex; 8, placenta; 9, spleen; 10,
cerebellum; 11, uterus; 12, stomach;
13, ovary; 14, testis; 15, salivary
gland; 16, lung; 17, adrenal gland. B,
tissue lysates from grade II and grade III were analyzed by
immunoblotting for hnRNP K expression (upper panel) and,
subsequently, reprobed with a vinculin Ab as a loading control
(middle panel). Quantitation of the ratio of hnRNP K:
vinculin is shown in the bottom panel.
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Fig. 6.
Regulation of c-Myc expression by growth
factors and antireceptor antibodies. A, modulation of
c-Myc promoter activity by hnRNP K and HRG. MCF-7 cells were
transiently transfected with a DNA for luciferase drawn by
c-Myc promoter and hnRNP K or control (Con) DNA, and
luciferase activity was measured 24 h after transfection. Some
cultures were treated with HRG (30 nM) for 16 h before
lysis. Relative luciferase activity is shown in the bottom
panel. B, tumors cells were treated with C225 or
HCT (50 nM) for 16 h, and c-Myc mRNA
expression was measured by Northern hybridization. GAPDH,
glyceraldehyde-3-phosphate dehydrogenase. Results are representative of
two experiments.
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Fig. 7.
Effect of hnRNP K expression on the biology
of breast cancer cells. A, Western blot analysis of
control and T7-tagged hnRNP K clones by anti-T7 mAb. The blot was
reprobed with an anti-vinculin Ab, as a loading control, and,
subsequently, with an anti-cMyc mAb. B, effect of T7-hnRNP K
expression on the activity of the c-Myc promoter-luciferase.
C, effect of T7-hnRNP K expression on the growth rate of
cells by MTT assay. D, effect of T7-hnRNP K expression on
anchorage-independent growth of MCF-7 cells. E,
representative photographs of soft agar colonies (n = 3).
-mediated activation of the agp gene involved in
the acute phase response (43) and induction of programmed cell death in
imaginal discs of Drosophila (44). These observations
suggest that the hnRNP K may differently influence cellular functions
in a cell type-specific manner.
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ACKNOWLEDGEMENTS |
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We thank Genentech Inc. for anti-HER2 antibody and Imclone Systems, Inc. for C225.
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FOOTNOTES |
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* This work was supported by the National Institutes of Health Grants CA80066 and CA65746, by the Breast Research program of The University of Texas M. D. Anderson Cancer Center, and by Bristol-Myers-Squibb Research Funds (to R. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Unidade de Oncologia-Hospital de Santa Maria,
Lisbon 1500, Portugal.
§ Member of the Board of Directors of Imclone Systems, Inc. and has stock options.
¶ To whom correspondence should be addressed. E-mail: rkumar@mdanderson.org.
Published, JBC Papers in Press, December 19, 2000, DOI 10.1074/jbc.M008514200
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ABBREVIATIONS |
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The abbreviations used are:
HER, human epidermal growth factor
receptor;
HRG, heregulin-1;
EGF, epidermal growth factor;
EGFR, EGF receptor;
C225, anti-EGF receptor antibody;
mAb, monoclonal
antibody;
HCT, anti-HER2 recombinant-derived humanized mAb. hnRNP K,
heterogeneous nuclear ribonucleoprotein K;
GDA, gene discovery array;
KH, K homology;
PAGE, polyacrylamide gel electrophoresis;
TGF, transforming growth factor.
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REFERENCES |
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