Vol.
6,
1097-1102,
September
1995
Cell
Growth
& Differentiation
Regulation
of Ornithine
Decarboxylase
mRNA Levels in
Human Breast Cancer Cells: Pattern of Expression and
Involvement
of Core Enhancer Promoter
Element
Paul S. Wright,’
Judith
Doreen
E. Cross-Doersen,
Paula A. Chmielewski,
Karen
A. Streng,
Marion
Merrell
Richard
and
Dow
R. Cooper,
Jerry A. Miller,2
1. Wagner,
Margaret
Research
A. Flanagan3
Institute,
Cincinnati,
Ohio
4521
5-6300
Abstract
Ornithine
decarboxylase
by growth
factors
through
the cell
cycle
this study, a variant
sequenced,
and
breast tumor
elevated
tumors
(ER)
in a wide
human
tumors
of cell types.
cDNA
mRNA
levels
in human
ODC
mRNA
was
as assessed by quantitative
analysis
The pattern
xenografts
In
was identified,
in estrogen
receptor-negative
(ER1
) when compared
with ER-positive
(MCF-7),
experiments.
is increased
for progression
variety
ODC
used to probe
about 3-fold
(MDA-MB-231
(ER)
expression
cell lines and xenografts.
autoradiographic
231
(ODC)
and is obligatory
of in situ hybridization
of ODC
was
mRNA
polarized
in MDA-MB-
to the
extreme
periphery of the tumor, whereas the distribution
of ODC
mRNA was more evenly distributed
in MCF-7 (ER)
xenografts.
staining
have
growth
This
patterns,
correlates
a differential
factor
in MDA-MB-231
in cell culture.
with
suggesting
hematoxylin
dependence
supply.
eosin
xenografts
on host vasculature
for
ODC
mRNA was elevated 5-fold
cells versus MCF-7 cells when analyzed
These relative mRNA levels correlate
with increased
levels of “core”
proteins
in MDA-MB-231
cells
MCF-7
cells.
Introduction
ODC4 is one
and
that ER4 and ER
enhancer
over that
of the rate-limiting
enzymes
binding
detected
Results
Isolation
nuclear
in
in the biosyn-
thetic
pathway
for polyamines,
ubiquitous
cellular
components essential
for protein
biosynthesis
and DNA replication
(i ). Progression
through
the cell cycle requires
biosynthesis
ofthe
pobyamines
putrescine,
spermidine,
and spermine
(2,
3). Elevation
of ODC
tissues after treatment
at the transcriptional,
levels (1 , 4). Elevated
acteristic
of cancer
Received
4/18/95;
t To whom
requests
Research
2 Present
Institute,
address:
ter, NY 14623.
3 Present
address:
02142.
4 The
abbreviations
receptor
positive;
hyde-3-phosphate
assay.
activity
is an early
event
in cells
or
with mitogens
and is tightly
regulated
translational,
and posttranslational
ODC and pobyamine
bevels are charcells of nearly
all types
(5). Several
revised
5/30/95;
accepted
6/23/95.
for reprints
should
be addressed,
at Marion
Merrell
Dow
2110
East Galbraith
Road, Cincinnati,
OH 45215-6300.
Department
of Biology,
Fisons Pharmaceuticals,
RochesOncogene
recent
reports
have linked
overexpression
of ODC
to malignant
transformation
of cells (6-9).
Critical
upstream
regulatory
elements
have been identified for transcriptional
control
ofthe
human
ODC gene (i 0,
i 1 ). Using nuclear
extracts,
it was found that protein
binding to the promoter
was more complex
with transformed
cells than in normal
diploid
fibroblasts
and that this correlated with the level of ODC
transcription
(i 0). These data
suggest that the elevated
expression
of ODC in transformed
cells
is regulated
in part by qualitative
and quantitative
usage of regulatory
elements
in the ODC
promoter.
Previousby, the relative
levels of ODC mRNA
and gene amplification
in various
human
breast cancer
cell lines have been
examined
in vitro (1 2). In this study, we compare
ODC gene
transcript
levels
in two types of breast
cancer
cells,
ER
MCF-7
cells and ER- MDA-MB-23i
cells in vitro and in
vivo. ODC mRNAs were increased
3- and 5-fold in MDAMB-231
xenografts
and cells, respectively,
when compared
with MCF-7
xenografts
and cells. Significant
increases
in
nuclear
proteins
interacting
with the “core”
enhancer
sequence
were detected
in MDA-MB-23i
cells when
compared with MCF-7
cells. All other potential
upstream
regulatory
sequences
tested,
including
an Myc
binding
site,
were unchanged
with regard to binding
of nuclear
proteins
extracted
from these two breast cancer
cell lines.
Science,
used are: ODC,
ER,
ER negative;
dehydrogenase;
Inc.,
80 Rogers
Street,
Cambridge,
MA
ornithine
decarboxylase;
ER,
estrogen
nt, nucleotide(s);
GAPDH,
glyceraldeEMSA,
electrophoretic
mobility
shift
of a New Human ODC cDNA.
A cDNA encoding
human
ODC was isolated
from a human
HepG2
cell Agti i
library
probed
with a partial
human
cDNA,
pODCiO/2H
(1 3). The insert of the resulting
clone,
AhODC85,
was sequenced
(see EMBL accession
no. X55362
for sequence).
There are several differences
between
this sequence
and the
previously
reported
cDNA,
as well as with the four human
genomic
clones
that have been reported
(14-17).
The sequence
reported
here has T replacing
C at nt -1 02, G
replacing
A at nt -39, and the deletion
of GGC at nt i 725.
These differences
do not alter the sequence
of the encoded
protein
because
they are in the 5’ and 3’ untranslated
regions.
The 5’ end of this clone
is 34 bp 3’ to the transcriptionab
start site. The 1 800-bp
EcoRI fragment
of the
human
ODC cDNA
was hybridized
with mRNA
prepared
from both MCF-7 and MDA-MB-23i
cells, yielding
a 2.2-kb
band on Northern
blots (Fig. iA). This was identical
to the
size band detected
with pODCiO/2H
(10, i3). The relative
bevels of ODC mRNAs
were 5-fold higher
in MDA-MB-231
cells when
compared
with MCF-7
cells and corrected
for
GAPDH
mRNA
levels in each cell type (Fig. 1 , B and C).
In Situ Hybridization
in Human
Breast Tumor
Xenografts.
The 1 800-bp
EcoRI fragment
of the human
ODC
cDNA
was also used as a template
for synthesis
of cRNA
probes
for in situ hybridization
analysis
of ODC
mRNA
levels in breast tumors.
Emulsion
autoradiographs
from in
situ hybridization
experiments
showed
high levels of ODC
mRNA
in the breast cancer
cells and not in host-derived
1097
1098
ODC
Regulation
A
in Breast
Cancer
..
Cells
2
1
4.4
-*.
2.4
-
3
4
#{149}
“:-
1.4-0.24-k
B
1
4.4
2
3
4
-k.
2.41 .4
-
:t
0.24-*
C
Analysis of Differences
in Nuclear Protein-DNA
actions with ODC Upstream
Promoter
Elements.
MCF-7
10000
-
MDAMB231
C,)
C
ci
E
;
were subtracted
from gray values
for adjacent
sections
hybridized
with antisense
probes
(specific
signal,
see “Materials and Methods”).
The specific
signal image for an MDAMB-23i
tumor
hybridized
with
ODC
cRNA
probes
is
shown
in Fig. 3. It can be seen that the spatial
pattern
of
ODC mRNA
accumulation
in these tumors
is concentrated
at the periphery
of the tumors.
MCF-7
xenografts
have a
more even distribution
of ODC mRNA
throughout
the section
(Fig. 4). This pattern
of niRNA
levels
parallels
the
degree
of necrosis
in the two xenograft
types
based
on
hematoxylin
and eosin staining
patterns
(data not shown).
Other
MCF-7
and MDA-MB-231
xenografts
were analyzed in this manner
for ODC mRNA
(Table 1 ) with similar
quantitative
and qualitative
results.
It is shown
in Table
i
that ODC
mRNA
levels in the outer regions
of the tumor
sections
were approximately
3-fold higher
in MDA-MB-231
tumors than in MCF-7 tumors,
while
inner hybridization
signals were about one-third
lower. The calculated
ratio of outer
to inner expression
was a net of approximately
4-fold higher in
the MDA-MB-231
tumors.
The mean value of the region of
highest
signal, the hot spot, was also about 3-fold
higher
in
MDA-MB-23i
xenografts.
This difference
in spatial
distribution
holds for other ER and ER- xenograft
models
as
well.
For instance,
ZR-75
tumors
(ER#{176}),like MCF-7,
have
an even distribution
of ODC mRNAs,
whereas
MDA-MB435 and MDA-MB-468
tumors
(ER)
exhibit
the extreme
peripheral
expression
as that shown
in MDA-MB-23i
tumors
(data not shown).
5000
10
RNNlane
20
(.tg)
Fig. 1. Northern
blot analysis
of ODC
and GAPDH
mRNAs
from MCF-7
and MDA-MB-231
cells. Total RNA was prepared
from MCF-7
(Lanes
1 and
2) and MDA-MB-231
(Lanes
3 and 4) cultures
and hybridized
with
32Plabeled
cDNA
probes
for ODC
(A) and GAPDH
(B) as described
in “Materials and Methods.”
Shown
are images
from phosphorimager
files (Lanes
1
and 3, 10 pg total RNA;
Lanes 2 and 4, 20 pg total RNA( used for quantitation.
C, volume
units for ODC
mRNA
corrected
for GAPDH
levels in each
cell type.
Inter-
Several
potential
regulatory
DNA sequence
elements
in the human
ODC
upstream
region
were tested by EMSA with nuclear
proteins
from MDA-MB-23i
and MCF-7
cells (see “Materiabs and Methods”
for obigonucleotide
sequences).
Only
the nuclear
proteins
binding
to footprint
lB were significantly
increased
in MDA-MB-231
cells over MCF-7
cells
(Fig. 5, upper
pane!).
The increase
in the upper
specific
complex
was 2.6-fold
based on scanning
densitometry.
This
upstream
element
includes
the consensus
“core”
enhancer
DNA sequence
common
to several
normal
and viral genes.
Other footprint
regions
examined
were footprints
VI (Fig. 5,
midd!e
pane!),
IA, IC, VII, or VIII (data not shown).
Additionally,
we tested the relative
levels of protein
binding
to
the ODC Myc binding
site found
in the first intron
(MB-i;
Fig. 5, lowerpane!).
No cell-specific
differences
were detected
in these other footprint
regions.
EMSAs performed
with MB-i
on extracts prepared
from dipboid fibroblasts
(IMR-90)
yielded
three major
complexes
in G0 and G1 phase cells (Fig. 6).
Nuclear
extracts from log-phase
fibroblasts
contained
only the
upper MB-i
complex,
which
was similar
in mobility
to the
complex
detected
in the bog-phase
breast cancer cells. Incubation
of the IMR9O log-phase
nuclear
extracts
with mAbs
directed
against c-myc protein
caused about a 50% decrease
in the upper MB-i
complex
(data not shown).
Discussion
stromab cells, based on the relative
number
of silver grains
(Fig. 2). Clusters
of cells in the MDA-MB-23i
tumors
had
high bevels of ODC
mRNA,
whereas
the distribution
of
grains in the MCF-7
tumors
was more uniform.
To analyze
mRNA
bevels in the two tumor types, we used
a combination
of quantitative
autoradiography
and image
analysis
(18). Calibrated,
linearized
gray values
for tissue
sections
probed
with the sense probes
(nonspecific
signal)
In this report,
we have shown
that tumor
levels of ODC
mRNAs
were elevated
in xenografts
of MDA-MB-23i
(ER-)
versus MCF-7
(ER)
cells using quantitative
imaging
of in
situ hybridization
in tumor
sections.
ODC
mRNA
levels
were
highest
near the extreme
periphery
of the MDAMB-231
tumors
(Fig. 4; Table
1). These cell-specific
differences
were
also evident
in vitro as shown
here and
previously
by others
(Ref. 12; Fig. 2). The ODC
enzyme
Cell
A
A Differentiation
B
.
4..
D
C
.
,
.-
Growth
,‘-.--.-
---
‘,
breast
and
tumor xenogratts.
Methods.”
II and
...
a
24
:
,
#{149}.
,
Fig. 2.
In situ hybridization
with ODC rnRNAs
in human
were processed
and hybridized
as describer)
in “Materials
50 pm.
12
I
.
Li,,
36
48
I
I
7
L
Fig. 3.
A digitized
image
of the specific
hybridization
for ODC
in an
MDA-MB-231
tumor.
In situ hybridizations
were pertormed
with sections
from MDA-MB-231
xenogratts
and quantitatecl
as des ribed
in “Materials
and Methods.”
Shown
are the specific
signals
(DPM/mm2
antisense
probe DPM/mnY
sense prol)o’( across
the tumor.
The scale br specific
hybridizalion ( DPM/mm2(
is given at the bottom
ot the tigure.
Tissue sections
from MCF-7
(A and B) and MDA-MB-231
(Cand
0) xenogratts
0, darkfield
images
of the tumor
regions
shown
in A and C (hrighttield(.
Bar,
activity
measured
in extracts
is approximately
the same
in these
two
breast
cancer
cell
lines,
suggesting
that
posttranscriptionab
mechanisms
significantly
contribute
to ODC
expression
(12).
The different
spatial
patterns
of ODC mRNAs
in the two
tumor
types likely
reflect
the relative
dependence
of ER
and ER xenografts
on host vasculature
for growth
factor
supply.
MCF-7
xenografts,
in animals
supplemented
with
estradiol,
do not develop
large necrotic
areas like MDAMB-231
xenografts,
based on hematoxybin
and eosin staining patterns.
Possibly
ER xenografts
are not as dependent
on paracrine
growth
factors due to production
of autocrine
factors
in response
to estrogens.
ODC expression
and polyamine synthesis
can be induced
by estradiol
in MCF-7
cells
(19).
Polyamines
and estrogens
can additively
stimulate
MCF-7
cell growth
in vitro (20, 2i). Additionally,
a link
between
ODC
expression
and angiogenesis
has been established
by other
investigators.
For instance,
DFMO,
an
irreversible
inhibitor
of ornithine
decarboxylase
(22), can
inhibit
tumor-induced
angiogenesis
in chick embryo
chorioallantoic
membrane
assays
(23).
The
polyamines,
spermidine
and spermine,
can stimulate
angiogenesis
in
chick
embryo
yolk-sac
membranes
(24).
Previously,
select
DNA
sequence
elements
in the 5’flanking
region ofthe
human
ODCgene
were implicated
in
the differential
transcription
of the gene in normal
fibroblasts
and transformed
cells
(10). The bevels of nuclear
proteins binding to all but one upstream
regulatory se-
1099
1100
ODC
Regulation
in Breast
Cancer
Cells
1
II
2
II
,
3
4
56
II
II
:‘C
‘4
‘4
Core
4.5
9.0
IS.e
13.5
22.5
‘
Fig. 4.
A digitized
image of the specific
hybridization
for ODC
in an MCF-7
tumor.
in situ hybridizations
were performed
with sections
from MCF-7
Xenografts
and quantitated
as described
in “Materials
and Methods.”
Shown
are
the specific
signals (DPtvVmm2
antisense
probe - DPPWmm2
sense probe) across
the tumor. The scalefor
specific
hybridization
(DPM/mm2)
is given atthe bottom
of the figure.
Table
tumors
1
Arrow,
the tumor
Quantitation
of ODC
region
of highest
mRNA
levels
signal
in MCF-7
(mean
,
.
.
FP VI
intensity.
and
MDA-MB-231
Paraffin
sections
of tumors
were hybridized
with cRNA
probes
to ODC,
and the mRNA
levels
were
quantitated
as described
in “Materials
and
Methods.”
The DPtvVmm2
are shown
for the inner portion
(-0.7-cm
diameter circle
in a 1 .0-cm diameter
tumor section),
the remaining
outer portion,
and the region of highest
signal (hot spot) for each tumor.
Mean values were
obtained
from triplicate
adjacent
sections
averaged
for three different
MCF-7
and MDA-MB-231
tumors.
M/2
‘
‘4
MB1
± SD)
Tumor
Outer
MCF-7
6.4 ± 3.7
Inner
2.7 ± 1 .9
Hot
spot
1 3.6 ± 9.3
Ratio
(out/in(
2.4
.
MDA-MB-231
18.9
± 9.3
1.8 ± 1.7
42.9
± 14.5
10.5
quences
shown
to differ
between
normal
fibrobbasts
and
transformed
cells were essentially
the same for MCF-7
and
MDA-MB-23i
cells (Fig. 6). The major difference
found was
in the EMSA performed
with FPIB (10), an oligonucleotide
containing
a “viral
core” enhancer
element
(nt -72 to -65;
5’-CTGGTTTG-3’;
Ref. 25). The nuclear
proteins
....-,.
that bind to
this obigonucleotide
were increased
in MDA-MB-23i
cells
when
compared
with
nuclear
proteins
from MCF-7
cells.
These data suggest that increased
occupancy
of this enhancer
sequence
may be involved
in establishing
the elevated
ODC
transcript
levels in MDA-MB-231
over MCF-7 cells.
It was also shown that the nuclear
proteins
binding
to the
upstream
Myc binding
site were unchanged
in the extracts
tested.
This negative
result
is of interest
as the murine
ornithine
decarboxylase
gene has been shown
to be transactivated
by c-myc (26), and c-myc
protein
bevels are higher
in MDA-MB-23i
cells than MCF-7
cells (Ref. 27 and data
not shown).
The sequence
containing
the Myc binding
site,
MB-i , was able to detect
qualitative
differences
between
extracts
prepared
from bog-phase,
growth-arrested,
and serum-stimulated
IMR9O
human
dipboid
fibroblasts
(Fig. 6).
This suggests
that nuclear
factors
interacting
with
MB-i
were sensitive
to differences
in the growth
state of normal
Fig. 5.
MCF-7 and MDA-MB-231
nuclear
protein
interactions
with ODC transcriptional
regulatory
elements.
Nuclear
extracts
were prepared
from duplicate
MCF-7
and MDA-MB-231
cultures
and used in EMSAs as described
in “Materials and Methods.”
The indicated
double-stranded
oligonucleotide
probes (notation
to the left of each panel)
were specifically
competed
with
unlabeled
oligonucleotides.
The lanes represent
MCF-7
nuclear
extracts
plus: ‘2P-labeled
oligonucleotides
(Lane
1), ‘2P-Iabeled
oligonucleotides
plus 100-fold
molar
excess cold competitors
(Lane 2). or no oligonucleotide
(one extract;
Lane 3); or
MDA-MB-231
nuclear
extracts
plus:
‘2P-labeled
oligonucleotides
(Lane 4).
‘2Plaboled
oligonucleotides
plus 1 00-fold
excess cold competitors
(Lane 5). or
no oligonucleotides
(one extract;
Lane 6). Arrowheads
to the right. the position
of saturable
complexes.
fibroblasts
during
and just after growth
inhibition.
The similarity
of gel shift patterns
with
extracts
from
bog-phase
fibrobbasts
to log-phase
MCF-7
and MDA-MB-23i
cell extracts suggests
little qualitative
or quantitative
difference
in
protein
binding
at MB-i
among
these cell types.
In summary,
it is shown
in this report that ODC mRNA
bevels were elevated
in xenografts
of ER MDA-MB-231
cells versus ER MCF-7
cells. There was also a difference
in
the spatial
distribution
of ODC mRNA
expression
between
the two tumor types. The quantitative
difference
may be due
in part to a difference
in the level of binding
of a nuclear
protein
at the core enhancer
element
found
65 nt upstream
from the ODC gene start site of transcription.
No evidence
Cell Growth
g0
log
Ii
2
3114
5
g1
6117
91
8
4
“4!
4
4
Fig. 6.
IMR-90
nuclear
protein
interactions
with MB-i . Nuclear
extracts
were prepared
from IMR-90
cells that were either continuously
incubated
in
medium
plus serum
(lo,’(.
serum
starved
for 24 Ii (g(.
or serum
starved
followed
I)y a 3-h incubation
in medium
plus serum
(g,(.
EMSAs
were
performed
with the MB-i
oligonucleotide
as described
in “Materials
and
Methods.”
All lanes contain
extracts
plus ‘2P-labeled
MB-i
oligonucleotide.
Lanes
2, 5, and 8 contain,
in addition,
a 100-fold
molar
excess
ot cold
competitor.
was found
for a role of c-myc
in elevated
expression
of
ODC mRNA
in MDA-MB-231
cells. The difference
in spatial distribution
of ODC
mRNA
in the xenografts
likely
reflects
the relative
dependence
of ER and ER- xenografts
on host vasculature
for growth
factor supply.
Materials
Isolation
and Methods
and Sequencing
of a Variant
Human
ODC
cDNA.
A cDNA encoding
human ODC (HSODC1
) was isolated from
a human
HepG2
cell Agti 1 library (Cbontech)
probed
with a
partial
human
ODC
cDNA,
pODC1O/2H
(13). Partial and
complete
EcoRl digestions
ofthe original
bacteriophage
clone,
AhODC8S,
generated
250-, 1 800-, and 2050-bp
fragments.
These fragments
were cloned
into the Bluescript
5K vector in
both orientations
(Stratagene).
The resulting
templates
were
sequenced
using double-stranded
DNA and T7 polymerase
(Pharmacia;
Ref. 28). The GC-rich
5’ end was sequenced
using single-stranded
template
in the absence and presence
of
deaza-dG
in order to resolve severe compressions.
Northern
Blot Hybridization.
Total RNA was prepared
from cells for Northern
blot analysis
using
a single-step
method
(29). RNAs were electrophoresed
in formaldehydedenaturing
gels (1 .4%) w/v, agarose)
and transferred
to Hybond N (Amersham)
as described
(30). An RNA ladder was
used for estimation
of mRNA sizes (GIBCO-BRL).
The blots
were probed
with 32P-labeled
cDNA
fragments
of the human ODC gene (1 800-bp
fragment
of AhODC8S)
and the
rat GAPDH
gene (31). A phosphorimager
(Molecular
Dynamics)
was used to quantitate
specific
mRNA
hybridizations.
Cell Culture and Tumor Xenografts.
MCF-7
(ATCC HTB
22) and MDA-MB-231
(ATCC HTB 26) were obtained
from
the American
Type
Culture
Collection
(Rockville,
MD).
Both cell lines were maintained
in improved
MEM (Bioflu-
A Differentiation
ids) supplemented
with
5 to 10%
fetal
bovine
serum
(GIBCO-BRL).
Tumor
xenografts
were produced
in female
nu/nu
athymic
nude
mice
(Harlan)
by injecting
tumor
pieces
(i to 2 mm3 ) with a trocar
near the mammary
fat
pads of the mice.
Estradiob
pellets
(Innovative
Research
of
America)
were implanted
in animals
carrying
the MCF-7
xenografts
to support
growth
of the tumors.
IMR-90
cells
were obtained
from the Corieb Institute
of Medical
Research
(Camden,
NJ) and cultured
in MEM
supplemented
with
antibiotics,
2 mtvi glutamine,
and 10% fetal bovine
serum.
In Situ Hybridization.
Tumors
were removed
from the
host animals
and fixed in ice-cold
paraformabdehyde
(4%,
overnight),
then embedded
in paraffin.
Sections
(5 to 6 pm)
were cut and mounted
on 3-aminopropyl
triethoxysibane
(Sigma Chemical
Co.)-treated
slides. In vitro transcription
of the
ODC
1800-bp
cDNA
was performed
using the Riboprobe
Gemini
lb system
(Promega).
T7 and T3 RNA pobymerases
were used to generate
the antisense
and sense cRNA probes
from
linearized
DNA
templates.
[35SjUridine
5’-(cs-thio)
triphosphate
(1 000-1 500 Ci/mmol;
Dupont-NEN)
was substituted for UTP to prepare the labeled
probes. !n situ hybridizations were performed
essentially
as described
by Simmons
et
a!. (32) with some modifications
(1 8). Briefly, the sections
were
dewaxed,
hydrated
in decreasing
ethanol
solutions,
postfixed
in 4% paraformaldehyde,
digested with proteinase
K (20 pg/mb
for 20 mm), refixed
in 4% paraformaldehyde,
treated
with
O.25%
acetic anhydride,
and dehydrated
in graded ethanol
washes. The hybridization
mixture
contained
50% deionized
formamide,
0.3 M NaCb, 20 m Tris-HCI
(pH 8.0), 10% dextran sulfate, 0.5 mg/mb yeast RNA, 5 m EDTA, 1 0 m sodium
phosphate,
20 msa DII,
plus approximately
300,000
CPM/pl
of either the sense or antisense
cRNA probes. Tissue sections
were hybridized
overnight
at 55#{176}C,
then washed
as follows:
(a) 5X SSC [0.15 M NaCI, 15 mii sodium
citrate (pH 7.0)1-10
mM DTT at 55#{176}C
for 30 mm; (b) 50’Y0 formamide,
2 X SSC, and
1 0 mM DTT at 65#{176}C
for 30 mm; (c) RNase A 120 pg/mI in 0.5
M NaCI,
1 0 mM Tris-HCI
(pH 8.0), and 5 ma EDTAI at 37#{176}C
for
30 mm; (d) repeat step (b) wash; and (e) 2X SSC, then 0.1 X
SSC for 1 5 mm each at room temperature.
The slides were
dehydrated
in the presence
of 0.3 s ammonium
acetate. Autoradiography
was performed
using XAR film (Kodak) or
labeled
Hyperfibm
(Amersham).
Emulsion
autoradiography
was also performed
using NTB-2
(Kodak).
The slides were
lightly stained with tobuidine
blue (0.02%,
30 5). Photomicrographs (bight and darkfield)
were taken with an Olympus
BH-2
microscope.
Quantitative
Imaging.
Plastic
or tissue
standards
contaming
known
amounts
of ‘4C were placed
in film cassettes
with
slides
containing
sectioned
tissue
and exposed
to
Hyperfibm.
After 1 or 2 days exposure,
the film was developed in Dektol
developer
(Kodak,
Rochester,
NY). The autoradiograms
were digitized
using a C-Imaging
1 280 computerized image analysis
system (Compix,
Inc., Mars, PA), and a
calibration
curve was used to convert
absorbance
to DPM/
mm2 (33, 34). Various
regions were outlined,
and the density
of the probe was measured.
Images of specific
labeling
were
generated
by using the calibrated
linearized
images and subtracting
the nonspecific
hybridization
(hybridization
using the
sense probe) from the total hybridization
(using the antisense
probe) as described
previously
(18).
EMSA. Nuclear
extracts
were
prepared
from cultured
cells as described
previously
(10, 35). Five pmol
of each
single-stranded
oligonucleotide
pair were end labeled
with
200 pCi of [y-t2PIATP
(3000 Ci/mmob;
Dupont-NEN)
and 10
units ofT4 polynucleotide
kinase (GIBCO-BRL).
After removal
1101
1102
ODC
Regulation
in Breast
Cancer
Cells
of unincorporated
nucleotides
by spin-chromatography
(Quick Spin G-25; Boehringer
Mannheim),
the single strands
were annealed for 10 mm at 85#{176}C,
followed
by slow cooling
(>3 h) to room temperature.
Specific activity as assessed by
liquid scintillation
counting was 0.5 to 2.0 X i0 CPM/pmol.
For each reaction, 0.01 pmol of probe was added to 8.5 p1 of
incubation
buffer (Stratagene), 4-8 pg nuclear extract, and 1
p1 unlabeled
competitor
DNA or H2O in a total volume
of
12.5 p1. The reactions were incubated at room temperature
for
nucleotide
sequence
gene. Gene (Amst.),
18. Wright,
Bitonti,
A.
P. 5., Cross-Doersen,
J., and Miller,
J. A.
mm. Prior to loading
on a 8.0 x 8.0 x 0.i cm 6% actylamide-0.5X
TBE gel (1 x TBE = 0.089
M Tris-borate,
089 M
xenografts
with
boric acid, and 0.02 M EDTA), 1 ml of 0.1% bromophenol
blue was added to each sample. The nucleoprotein
complexes
were electrophoresed
at room temperature
in 0.5X TBE at 20
mA until the dye front had migrated 5 cm. The gels were dried
and exposed to X-OMAT
X-ray film with Cronex (Dupont-
1 9. Thomas,
T., and Thomas,
mRNA,
enzyme
activity,
and
30
NEN)
upper
intensifying
strand ofthe
screens
at -80#{176}C.The sequences
of
oligonucleotides
tested were as follows
from Ref. 26, all others from Ref. 10): (a) Core (FP lB),
CCGATCGTGGCTGGT1TGAGCTGGTGC-3’;
(b)
FP
5
1
MB-i
, 5 ‘-CGCCGCACACGTGCCCGGGGC-3
‘ ;
(d)
IA, 5
‘ ; (e)
IC, 5’-TCCCGGCCGGAA-3’;
( f) FP VII, 5’-GCGCGGAC-
the
CAGTTCCAGGCGGGCGAGA-3’;
5’-
and
(g)
FP VIII,
1(c)
5’VI,
(c)
FP
FP
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