Rapid Inhibition of c-myc Gene
Expression by a Glucocorticoid
in the Avian Oviduct
Charles Rories*, Chu Kwan Lau, Karen Fink, and
Thomas C. Spelsberg
Department of Biochemistry and Molecular Biology
Mayo Clinic
Rochester, Minnesota 55905
Hormone Action and Oncogenesis Section (C.R.)
National Cancer Institute
Bethesda, Maryland 20892
The glucocorticoid dexamethasone (DEX) causes a
rapid, reversible reduction in c-myc mRNA level in
the oviducts of estrogen-treated, immature chickens. The c-myc mRNA level begins to decrease by
5 min after injection of 0.5 mg DEX, reaches a
minimum of 10% of the control value by 30 min, and
returns to 30-40% of the control value by 4 h post
injection. This rapid effect of DEX on the c-myc
mRNA level occurs in both diethylstilbestrol-stimulated and diethylstilbestrol-withdrawn oviducts. The
effect is dose dependent, with reduction of the cmyc mRNA measured with as little as 10 ^g DEX
injection (0.03 jug/g BW). The effect of the steroid is
gene specific with H2B histone mRNA displaying a
significantly reduced response. The effect is also
tissue specific with liver displaying an increase of
170% of control values in c-myc mRNA level by 30
min after injection of 0.5 mg DEX. The reduction of
avian oviduct c-myc mRNA levels by DEX may play
a role in glucocorticoid inhibition of cell proliferation
in this tissue. The rapidity of the steroid effects on
c-myc expression makes it likely that the steroidinduced reduction of c-myc mRNA levels represents
a direct primary action of the steroid-receptor complex on the c-myc gene expression. (Molecular Endocrinology 3: 991-1001, 1989)
can also regulate gene expression by regulating the
rates of degradation of specific mRNAs (5-7) and protein synthesis and processing (1). These steroid-induced changes in the mRNA levels of specific genes
occur at both early (15-20 min) and late (2-6 h) periods
post injection, and their magnitude and timing is specific
for both the hormone and the particular target cell (1,
3-5).
A few steroid-sensitive genes have been shown to
increase their steady state mRNA levels within 15 min
after steroid administration. Examples of such early
genes are those of the mouse mammary tumor virus
(8), the early induced genes of rat uterus (9, 10), and
the pS2 gene of human breast cancer cell line MCF-7
(11). However, the vast majority of studies of the molecular mechanisms by which steroid hormones regulate genetic expression involved late genes whose transcription rates and mRNA levels are not measurably
altered until 2 or more hours after hormone injection.
Good examples of such late genes in the avian oviduct
are the egg white protein genes, e.g. ovalbumin, ovomucoid, and lysozyme (4,12-15). There is a 2- to 3-h
lag period between the time steroids are administered
to estrogen-withdrawn chickens, and the detectable
significant increases in the rate of transcription and
mRNA levels for these genes. Other examples of late
genes include glucocorticoid induction of the rat hepatic
a2u-globulin gene (16), and estradiol induction of the
liver vitellogenin gene (17) whereby the significant increases in transcription and mRNA levels of these
genes occurring 90 min or longer after steroid administration.
Early studies using nuclear run-off transcriptional
analyses to assess changes in steroid-regulated RNA
polymerase II activities have shown two distinct phases
of increases in transcriptional activity, with a sharp peak
occurring immediately after steroid administration (early
response), and a broader peak occurring over the
period between 2 and 12 h after steroid administration
(late response) (14, 18). The timing of the above described early and late transcriptional responses to ste-
INTRODUCTION
The specific molecular mechanism(s) by which steroids
exert control of gene expression and cell proliferation
is not known. Steroids bind to their receptors which, in
turn, bind to the nuclear acceptor sites within minutes
after injection into an organism (1, 2). The result of this
nuclear binding is the alteration of the rates of transcription of specific genes (1, 3-5). Active steroid receptors
0888-8809/89/0991 -1001 $02.00/0
Molecular Endocrinology
Copyright © 1989 by The Endocrine Society
991
MOL ENDO-1989
992
roids coincides, respectively, with these two peaks of
steroid-induced RNA polymerase II activity. These data
support the early and late gene response model, which
encompasses a more general, biphasic response of
genetic activity to steroid hormone action,. The large
interval of time which elapses between steroid administration and the measurable changes in transcription
of the early and late genes suggests that the transcriptional response to steroids is mediated by two (or more)
related but distinct mechanisms.
This laboratory has attempted to identify rapidly regulated, steroid-sensitive genes in the avian oviduct
model that code for protein products which, functioning
as regulatory factors, mediate the steroid regulation of
the late responding genes. Emphasis was placed on
certain protooncogenes whose protein products are
found in the cell nucleus and suspected of having gene
regulatory functions. It was recently reported by this
laboratory that c-myc mRNA concentration in the avian
oviduct began to decrease within 5 min after administration of progesterone to estrogen-primed chickens,
reaching a minimum of 20% of control values at about
30 min, and returning to the control mRNA level by 4 h
post injection (19). This effect of progesterone on cmyc mRNA levels was found to be tissue specific and
dose dependent. Progesterone was found to also reduce c-myc mRNA concentration in oviducts of estrogen-withdrawn chickens with the same rapid kinetics
observed in estrogen-stimulated chicken oviducts (Fink,
K. L, T. C. Spelsberg, unpublished data). Since the
avian oviduct contains functional glucocorticoid receptors (20), and since the delayed response associated
with glucocorticoid induction of egg white protein gene
transcription is similar to that for progesterone induction
of these same genes (21), the effect of the glucocorticoid dexamethasone (DEX) on oviduct c-myc mRNA
levels was investigated.
This paper describes a rapid, tissue-specific decrease
in c-myc mRNA concentration, induced by glucocorticoids, in the oviducts of both estrogen-stimulated and
estrogen-withdrawn chickens. In the same animals,
glucocorticoid treatment causes a rapid increase in liver
c-myc mRNA levels. The data presented here suggests
that the control of the growth of the avian oviduct tissue
by steroid hormones may involve direct hormonal regulation of oviduct c-myc protooncogene expression.
RESULTS
In order to determine the interval of DEX regulation of
oviduct c-myc mRNA levels in both diethylstilbestrol
(DES)-stimulated and DES-withdrawn chickens, pairs
of animals were injected with 0.5 mg DEX and killed at
varying time points over a 4-h period. The oviducts,
livers, and spleens were quickly removed and frozen
on dry ice. The total oviduct RNAs were isolated from
the frozen tissues within 2 weeks of storage, and the
relative concentrations of c-myc mRNAs were deter-
Vol 3 No. 6
mined by Northern blot analysis using 32P-labeled vmyc cDNA as hybridization probe. Figure 1 represents
a Northern blot of total RNA isolated from DES-withdrawn chickens at various periods after DEX injection
at zero time. The blot was probed with [32P]-v-myc
cDNA to identify c-myc mRNA bands. To test for gene
specificity in the steroid regulation, some blots were
simultaneously incubated with [32P]-v-myc cDNA and
[32P]H2B (histone gene) cDNA. In other cases, the same
blot was washed to remove [32P]v-myc cDNA, and was
reprobed with [32P]H2B cDNA. As shown in Fig. 1, DEX
causes a rapid, reversible decrease in c-myc mRNA
levels in the oviducts of DES-withdrawn chickens. Figure 2 shows that DEX causes a similar rapid decrease
in c-myc mRNA levels in the oviducts of DES-stimulated
chickens. Injection of vehicle (propyleneglycol) alone
into both DES-stimulated and DES-withdrawn chicks
resulted in only minor changes in oviduct c-myc mRNA
levels (data not shown). Figures 1 and 2 also show that
the concentrations of the mRNA for H2B, a constitutive
gene which is not known to be regulated by steroids,
are not markedly changed after injection of the animals
with dexamethasone. These data support that the response of c-myc to DEX is gene specific and that the
estrogen status of the animals does not affect DEXinduced changes in the c-myc mRNA levels.
To further analyze these results, the optical absorption values of the autoradiograph bands were quantified
by scanning densitometry. All autoradiographs were
developed so that the most concentrated bands absorbed light below the upper thresholds of the film and
densitometer. The c-myc mRNA concentrations in the
oviducts from both DES-primed (Fig. 3A) and DESwithdrawn (Fig. 3B) animals displayed decreases as
early as 5 min after ip injection of 0.5 mg DEX, reached
a minimum value of less than 10% of the control (preinjection) level at 30 min, and then returned to approximately 50% of the control level by 4 h postinjection
(Fig. 3). Interestingly, vehicle shows an increase in cmyc mRNA in DES-withdrawn chicks during the first 2
h while the H2B mRNA levels show minimal changes
over that period (Fig. 3A). In DES-treated animals (Fig.
3B) the H2B mRNA shows an early steroid-induced
increase during the first 2 h. These changes in densitometric values were reproducibly demonstrated in
three to five separate animal experiments and support
the steroid dependency and gene specific effects of
DEX on c-myc mRNA levels.
The effect of DEX dosage on c-myc mRNA concentration in oviducts of DES-withdrawn chickens was then
examined by injecting various amounts of DEX and
analyzing the mRNA species at 40 min after injection
by Northern blotting (data not shown). Figure 4 shows
densitometric values whereby the rapid reduction in cmyc mRNA concentration occurs at a dose as low as
10 >iQ DEX (30 ng/kg BW) and achieves a minimum
{i.e. maximal effect) at 100 M9 DEX where a reduction
of c-myc reaches 10% of that of control values. These
dose-dependent changes also support the steroid requirement for the reduction in c-myc mRNA levels.
993
Glucocorticoid Reduces Oviduct c-myc mRNA
Dexamethasone Regulation of c-myc mRNA
Levels in Estrogen-Withdrawn Chicken Oviduct
o1
veh
5'
101
15'
30'
1 hr
2hr
4 hr
6 hr
c-myc
~ 2.3 kb
H2B
~ 0.6 kb
Fig. 1. Chronology of the DEX Regulation of c-myc mRNA Levels in the Oviduct from Estrogen-Withdrawn Chicks
DES-withdrawn chickens were injected (ip) with 500 ng DEX at zero time. Chickens were killed and oviducts removed at times
shown at the top of the figure. Total oviduct RNA (25-30 ^g) for each time point was resolved on a denaturing agarose gel, and
specific mRNA species were detected by Northern blot analysis as described in Materials and Methods. RNA size standards were
coelectrophoresed and stained. The size of c-myc and H2B mRNA determined by their mobilities in the gel are shown at right of
the figure. Oviduct c-myc mRNA was detected by hybridization with 32P-labeled \i-myc cDNA. H2B mRNA was detected by washing
the blot, followed by hybridization with 32P-labeled H2B cDNA.
Dexamethasone Regulation of c-myc mRNA
Levels in Estrogen-Stimulated Chicken Oviduct
o*
veh
51
10*
151
c-myc
H2B
30*
1 hr
2 hr
4 hr
6 hr
- 2.3 kb
~ 0.6 kb
Fig. 2. Chronology of the DEX Regulation of c-myc mRNA Levels in the Oviducts from Estrogen-Stimulated Chicks
The experiments were performed as described in the legend to Fig. 1 except that estrogen-stimulated oviducts were used.
The total cellular RNAs isolated from liver and spleen
of DEX-treated chickens, were also analyzed to determine the tissue specificity of DEX action on the c-myc
mRNA concentration. Figure 5A shows the densitometric results of [32P]v-myc cDNA hybridization to a
Northern blot of total avian liver RNA, isolated after
injection of 500 ng DEX into DES-withdrawn chickens.
An increase (150-200% of control values) in c-myc
mRNA levels from liver was consistently found by 20
to 25 min after DEX injection. Interestingly, the amount
of H2B mRNA is decreased after DEX treatment of the
animal in liver (Fig. 5A). As shown in Fig. 5B, when the
c-myc mRNA values are normalized to the H2B mRNA
values, a dramatic increase in the c-myc mRNA concentration is observed. In contrast to the rapid decline
of the c-myc mRNA in the oviduct, the c-myc mRNA
levels in spleen show a more gradual multiphasic decline to a minimum of 5% control at 4 h (Fig. 6). These
tissue specific responses demonstrate that the rapid
decrease in c-myc mRNA concentration observed in
MOL ENDO-1989
994
Vol 3 No. 6
<
2
E
100
200
300
400
300
400
Time (min.)
100
200
Time (min.)
Fig. 3. Densitometric Quantification of the Effects of DEX on c-myc mRNA Levels in the Oviducts from Both Estrogen-Stimulated
and Estrogen-Withdrawn Chicks
DES-stimulated and DES-withdrawn chickens were injected with 500 ^g DEX. Other DES-withdrawn animals were injected with
vehicle (propyleneglycol) only. After injection, the oviducts were removed at the times indicated. Total RNA was isolated and
analyzed by Northern blot analysis. The c-myc mRNA from DEX-treated (•) or vehicle-treated (•) animals was detected by
autoradiography of the 32P-labeled v-myc cDNA, and H2B mRNA (A) was detected by hybridization with H2B cDNA. The relative
amounts of c-myc mRNA at the various periods post injection were determined by scanning densitometry of the autoradiographs
of the Northern blots as described in Materials and Methods. The mean of values obtained from several densitometric scans of a
typical experiment are presented. A, Effects of DEX on c-myc mRNA levels in estrogen-withdrawn chicken oviduct; B, same effects
in estrogen-stimulated chicken oviducts.
the chick oviduct after DEX administration is not a
general effect of glucocorticoid on c-myc expression in
all tissues.
DISCUSSION
The data presented here show that the glucocorticoid,
DEX, causes a rapid, specific, 90% reduction of c-myc
mRNA concentration in the avian oviduct. The rapid
effect of DEX on c-myc gene expression is both dose
dependent, tissue specific, and gene specific. Of the
three tissues examined in this study, only oviduct cells
show a consistent, rapid decline in c-myc mRNA concentrations after DEX administration. The c-myc mRNA
levels in spleen show a biphasic, slower response in
the first half-hour after DEX treatment, while in liver
there is an increase in the c-myc mRNA concentration.
The increase in liver c-myc mRNA concentration after
995
Glucocorticoid Reduces Oviduct c-myc mRNA
Effects of Dexamethasone Dose (40 Min. post injection)
on c-Myc mRNA in the Oviducts from DES-wd Chicks
100—•— Myc
mRNA
\
- A - H2B
' 80-
\
A
•
>•-
Relaifive i \mount
o
60•
•
40-
\
.V\
20-
n
U
i
i
I
I
i
I
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Dexamethasone Dose (Mg)
Fig. 4. Densitometric Quantification of the Effects of DEX Dose Response on c-myc mRNA Levels in the Oviducts from DESWithdrawn Chicks (40 min post injection)
The animals were given increasing amounts (10 to 500 ^g) of DEX and then killed 40 min later. Northern blot analyses for both
c-myc (•) and H2B (•) mRNAs was performed as described in the legend to Fig. 1 and a densitometric analyses of these blots
was performed as described in the legend to Fig. 3. Details of the methods are described in Materials and Methods. The means of
values from several densitometric scans of a typical experiment are presented.
DEX treatment is enigmatic, as increased expression
of c-myc in liver is associated with liver cell proliferation
(22), and glucocorticoids reportedly inhibit proliferation
of liver cells (23). Liver tissue is comprised of cell
populations whose proliferations display a wide variation in sensitivity to glucocorticoids (24). The reproducible increase in c-myc mRNA levels observed in liver
after glucocorticoid administration may represent a
large increase in c-myc mRNA levels in a subpopulation
of cells. In any event, the DEX-induced increase in the
liver c-myc mRNA levels and the rapid, clearly defined
decrease of oviduct c-myc mRNA levels demonstrate
the tissue-specific nature of the steroid-regulated cmyc gene expression.
The gene specific response is demonstrated by the
minimal effects of DEX on H2B mRNA levels in the
oviduct, liver, and spleen. It should be mentioned that
the mRNA of some other control genes, actin, and
tubulin, did display more pronounced changes than the
H2B mRNA in response to DEX.
All measurements of chicken oviduct c-myc mRNA
levels show a degree of variability. About 30 min after
DEX injection, minimum values of oviduct c-myc mRNA
levels range between 5% and 20% of preinjection levels. By 4 h postinjection, the c-myc mRNA levels range
between 40% and 75% of preinjection levels. There is
also some variability in the minimum amount of DEX
needed to give partial reduction of c-myc mRNA levels.
Even the levels of the oviduct histone H2B mRNA vary
±20% during the first 4 h after DEX treatment. These
variabilities in oviduct c-myc mRNA levels is probably a
combination of effects from three different sources: 1)
experimental methodology error, due, for example, to
differences between chickens in the amount of steroid
actually injected into each animal, differences in the
amounts of RNA loaded per gel lane, efficiencies of
transfer of mRNA to the membrane, and efficiencies in
the hybridization of the 32P-labeled probes to the membrane-bound mRNA; 2) differences in the steroid metabolism between individual chickens, and whole body
disposition of the steroid which might produce differences in the relative effects on the amounts of specific
mRNA species in RNA isolated from different oviducts;
3) effects of circannual rhythms which have been shown
to exist for the weight, protein content, RNA polymerase II activity, and concentrations and activities of the
progesterone receptor in the avian oviduct (25,26). The
latter is probable since these studies were performed
throughout the year.
A rapid increase in c-myc gene expression is one of
the early events that occur when quiescent cells are
induced to proliferate by mitogens and growth factors
(27, 28). The steady state c-myc mRNA levels are
generally higher in proliferating cells than in quiescent
or differentiating cells (29-32). Enhanced c-myc gene
expression is associated with estrogen-induced cell
MOL ENDO-1989
996
Vol 3 No. 6
Effects of 0.5 mg Dexamethasone on c-Myc and H2B mRNA
Levels in DES-withdrawn Chicken Livers
175
150125100-
250
400
300-
200-
100-
150
175
200
225
250
Time (min.)
Fig. 5. Densitometric Quantification of the Effects of DEX on c-myc mRNA Levels in Liver Estrogen-Withdrawn Chicks
DES-withdrawn chicks were injected with 500 pg DEX at zero time. At each interval of time after injection, the chicks were killed
and the livers removed. The liver RNAs were analyzed by Northern blot methods and quantified by densitometry of the
autoradiographs as described previously in the legends to Figs. 1 and 3 and Materials and Methods. A, Relative amount of c-myc
(•) and H2B (A) mRNA in DES-withdrawn chicken liver. B, Same data from A as a ratio of the amount of c-myc mRNA to the
amount of H2B mRNA.
proliferation in the rat uterus (33, 34), in human breast
cancer cells (35), and in the immature chick oviduct
(36). In contrast, c-myc expression is rapidly reduced
in association with glucocorticoid inhibition of proliferation of murine P1798 thymic lymphosarcoma cells (37)
and S49 lymphoma cells (38). We report here that
glucocorticoids also inhibit c-myc expression in the
spleen which probably reflects the steroids effect on
lymphocyte cell proliferation. We also show that DEX
reduces c-myc mRNA levels in the avian oviduct, with
a magnitude and timing similar to the inhibitory effects
of progesterone on oviduct c-myc expression (19). Both
glucocorticoids and progesterone are known to inhibit
estrogen-induced proliferation of avian oviduct tissue
(39, 40), and progesterone promotes oviduct differentiation (41,42). Thus, the correlation between reduction
of the oviduct and spleen c-myc gene expression induced by glucocorticoids and progesterone and the
effects of these steroids on cell proliferation, suggests
that a direct regulation of c-myc expression by these
hormones may be one of the key events mediating
steroid control of whole animal tissue proliferation.
The reason for the 2- to 3-h delay in the response to
steroids by most steroid regulated structural genes,
Glucocorticoid Reduces Oviduct c-myc mRNA
997
Effects of 0.5 mg Dexamethasone on c-Myc and H2B mRNA
Levels in DES-Withdrawn Chicken Spleens
100-
80-
60-
40-
20-
250
100-
80-
60-
40-
20-
125
150
100
Time (min.)
175
200
225
250
Fig. 6. Densitometric Quantification of the Effects of DEX on c-myc mRNA Levels in Spleen from Estrogen-Withdrawn Chicks
These experiments were performed exactly like those in the legend to Fig. 5. The DES-withdrawn chicks were injected with 500
^g DEX for various times, and the animals were killed, and the spleens were removed. The total spleen RNAs were analyzed by
Northern blot methods and quantified by densitometry of the autoradiographs. A, Relative amount of c-myc (•) and H2B RNA (•)
with response to DEX time course. B, Same data as in A, but here the data are presented as the ratio of the amount of c-myc
mRNA to the amount of H2B mRNA.
termed late genes in this paper, is not known. A goal in
this laboratory has been to identify genes in the chicken
oviduct which undergo rapid change in transcription
rate immediately after steroid injection. Rapidly regulated oviduct genes which code for nuclear proteins
with possible regulatory functions would provide a
model system for analyzing the structural-functional
relationships between the steroid regulations of both
early and late gene expression. Support for such a
model is found in the midge fly system as outlined
elsewhere (1, 43, 44). The steroid ecdysone induces
the synthesis of early proteins which localize in the
nucleus at the chromosomal domains of late genes (45,
46). The discovery of early and late transcriptional
responses in midge salivary glands after administration
of ecdysone led to the proposal in those papers that
some of the steroid-responsive early genes code for
regulatory (e.g. transcription) factors which are required
for the transcription of the late genes (45, 46). Such a
cascade model might account for the biphasic transcriptional response to steroids observed in higher organisms mentioned earlier in this paper (1, 14, 18). The
functions of the steroid-induced early proteins that alter
expression of the late genes might include the large
MOL ENDO-1989
998
scale changes in chromatin ultrastructure (47, 48)
changes in DNA methylation (47-49), changes in chromatin structure associated with the appearance of
DNase I hypersensitive sites (50, 51), and finally, the
more recently reported regulation of the synthesis or
activation of transcription factors (1, 52-55).
The c-myc oncogene codes for a DNA-binding protein
that concentrates in the cell nucleus (56). While the
exact mechanism as to how the c-myc protein regulates
DNA replication is not known, there is evidence which
suggests that this mechanism is indirect (57) and that
the c-myc protein is involved in the regulation of other
genes (58-60). We have shown both in this paper and
elsewhere (19) that the oviduct c-myc mRNA levels
begin to decline within 5 min after injection of glucocorticoids and progestins, respectively. The action of glucocorticoids and progestins on c-myc gene expression
thus supports the cascade model of steroid action as
proposed previously by this laboratory (1, 43, 44). In
this model, the c-myc gene is a steroid-responsive early
gene which codes for a regulatory protein that can
modulate expression of the more commonly measured
late structural genes after a lag period (1, 43, 44).
Such a model could be used to address some of the
major questions relating to the mechanisms of steroid
hormone action such as: 1) the events occurring right
after the rapid association of steroid-activated receptors with specific acceptor sites on oviduct chromatin
(1, 45, 46, 61, 62), 2) the role of the steroid response
elements flanking steroid-responsive genes in binding
transcription factors and other regulatory gene products (1,43, 44, 52-55) as opposed to steroid receptors
(63-67), and 3) the role, if any, of changes in chromatin
structure and/or binding of transcription factors to the
gene's promoter region induced by steroids (47-53,
68, 69). The 5- to 10-min response of c-myc gene
expression to glucocorticoids and progestins suggests
that the steroid-activated receptors might interact directly with regulatory sites controlling c-myc mRNA
transcription, or if the rapid action of DEX is at the level
of the half-life of c-myc mRNA, then a whole new model
for steroid action might have to be considered. The
down-regulation of c-myc mRNA levels by agents that
inhibit growth and induce cell differentiation have been
shown to be regulated either at the transcriptional level
or at the posttranscriptional level, or both (29, 30, 70).
Experiments to determine the mechanism(s) by which
c-myc expression is inhibited by glucocorticoids and
progestins are in progress.
MATERIALS AND METHODS
Pretreatment of Chickens and DEX Administration
Chickens used in these experiments were Dekalb White Leghorn pullets received as 1-day-old hatchlings from a local
hatchery. At 1 week of age the animals were given daily 0.2
ml, sc injections of 1 mg DES in sesame oil. The chickens
were considered fully estrogen stimulated after receiving DES
5 days/week for at least 3 weeks. Estrogen-withdrawn birds
Vol 3 No. 6
represented fully stimulated chickens withdrawn from DES for
at least 3 weeks before use. DEX (Sigma, St. Louis, MO) was
dissolved in propyleneglycol at various concentrations to give
the desired amount to be injected in 0.2 ml, and was injected
ip.
Isolation of RNA after DEX Administration
In all experiments, chickens were injected with DEX at time
zero (designated 0) and a minimum of two chicks were killed
by cervical dislocation at each time point. Oviducts (magnum
portions), livers, and spleens were quickly excised and frozen
on dry ice. Frozen tissues were stored at - 7 0 C until time to
isolate RNA. RNA was isolated after homogenization of 0.9 g
tissue in 35 ml 6 M Guanidine-HCI, 20 ITIM Na-acetate, followed
by ultracentrifugation through a cushion of 8 ml 5.7 M CSCI,
0.1 M EDTA, pH 7.0, as described previously (71). RNA
concentration was determined by UV absorption at 260 nm
and using 0.04 mg/ml/U 260 nm absorbance (72). In some
experiments, RNA concentrations were confirmed by the Orcinol assay (73). Samples of each set of the RNA preparations
were first analyzed by glyoxal gel electrophoresis (74, 75),
followed by ethidium bromide staining, with visual inspection
under UV illumination to detect any degradation of the RNA
species. The method for the isolation of oviduct RNA resulted
in little or no sample degradation. However, the RNA preparations from chicken liver and spleen occasionally displayed
some degradation of the RNA species, requiring a repeat of
those experiments.
RNA Electrophoresis and Transfer to Filter
Total cellular RNA (25-30 ^g from each preparation) was
analyzed by glyoxal, 1 % agarose, gel electrophoresis to give
a size-dependent distribution of RNA molecules (74). One lane
of each slab gel was loaded with RNA molecular size standards
(BRL, Gaithersburg, MD). After electrophoresis, the gel lane
was removed, stained, and photographed. A plot of log (no.
of nucleotides) vs. position in the gel was made based on
mobility of these size standards, and the interpolation on this
plot was used to determine sizes of the mRNA bands. The
RNAs were transferred from gel to Magna 66 nylon membrane
(MSI, Fisher Scientific, Pittsburgh, PA) by capillary flow of 3 M
NaCI, 0.3 M trisodium citrate (20x SSC) as described for
transfer of DNA by Southern (74, 75). After transfer, the
membrane (i.e. the Northern blot) was marked to show the
position of the wells, so that mRNA band migration distance
could later be determined. The blot was air dried and then
baked 2 h at 80 C under vacuum to fix the RNA to the
membrane.
Hybridization Analysis of mRNA
Before the hybridization, the membrane was soaked for 5 min
in 3x SSC, 0.1% (wt/wt) sodium dodecyl sulfate (SDS), then
for 20 min in recently boiled 20 mM Tris, pH 8.0, and finally
again for 5 min in 3x SSC, 0.1% (wt/wt) SDS. The membrane
was then sealed in a bag with 15 ml freshly-made prehybridization solution [50% (vol/vol) deionized formamide, 5x Denhardt's solution (76, 77), 3x SSC, 0.01 mg/ml polyadenosine,
0.05 mg/ml denatured salmon sperm DNA, 0.1% (wt/wt) SDS,
and incubated at least 3 h in 43 C water bath with gentle
agitation.
The DNA hybridization probes used were linear DNA fragments labeled to specific activity of about 109 cpm//Ltg using
Multiprime DNA labeling kit (Amersham, Arlington Heights, IL)
and [32P]dCTP with a specific activity of approximately 3000
Ci/mmol (New England Nuclear Research Products, Boston,
MA). The c-myc mRNA was detected with a 1560 base pair
fragment of the v-myc gene of avian myelocytomatosis virus
MC29 (78, 79) which contains sequences homologous to
exons 2 and 3 of c-myc protooncogene, but has no homology
Glucocorticoid Reduces Oviduct c-myc mRNA
to the c-myc exon 1. The c-myc and H2B DNA (80) probes
were purchased from ONCOR (Gaithersburg, MD). All heatdenatured 32P-labeled DNA probes were mixed with 10 ml
fresh prehybridization solution to have activity of about 2 x
106 cpm/ml. After prehybridization, the solutions were replaced
with the radiolabeled probe solution, the bags were resealed,
and incubated at least 18 h in 43 C water bath with gentle
agitation. In some instances, both c-myc and H2B probes
were incubated simultaneously with the gel blot; in other
instances, only one probe was used. After hybridization, the
membranes were washed 10 min in 250 ml warm 3x SSC,
0.1% (wt/wt) SDS, then sealed in a bag with 60 ml 1 X SSC,
0.1% (wt/wt) SDS and agitated for 20 min in a 55 C water
bath. This was followed by two successive 20-min washes in
60 ml 0.5x SSC, 0.1% (wt/wt) SDS at 55 C. These washes
were of sufficiently high stringency that [32P]-v-myc probe
remained hybridized to only one mRNA species on the blot.
The membranes were autoradiographed with intensifying
screens for 5-12 h at - 7 0 C, using Kodak X-Omat AR film
(Eastman Kodak Co., Rochester, NY). Films were developed
by a Kodak X-Omat M20 film processor. In order to reprobe
the Northern blots with different cDNA probes, membranebound radiolabeled probes were removed by agitating the
bagged membranes in 60 ml 10 IDM Tris, pH 8.0, for 6-9 h at
65 C and the membranes incubated with different radiolabeled
probes as described above.
Densitometry of Autoradiograph mRNA Bands
The relative amounts of c-myc mRNA in the RNA preparations
were obtained by densitometric analysis of hybridization bands
in autoradiograph films. Autoradiographs were scanned with
a transmittance scanning densitometer (model GS 300, Hoefer
Instruments, San Francisco, CA) to give an absorbance (A)
profile across each mRNA band. Analog A absorption profiles
were digitized and peak areas were integrated using data
analysis software (Holter GS 360 data system) with an IBM
PC/XT computer (IBM Corp., Boca Raton, FL). All absorbance
values which were used in these studies resided within the
linear capacities of the densitometer using both standard
absorbance filters as well as titrated RNA preparations which
had been analyzed by Northern blots.
Acknowledgments
This research was accomplished with the technical assistance
of Ms. Deborah Halverson and Ms. Kay Rasmussen. The
authors would like to thank Ms. Jackie Keller and Ms. Sherry
Linander for their secretarial assistance.
Received June 30,1989. Revision received March 1,1989.
Accepted March 14,1989.
Address requests for reprints to: Dr. Thomas C. Spelsberg,
Department of Biochemistry and Molecular Biology, Mayo
Clinic, Rochester, Minnesota 55905.
This research was funded by Grant HD-9140-P1 from the
NICHHD and the Mayo Foundation.
* Supported by the NIH Reproductive Training Grant HD07108.
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