(Received for publication, October 22, 1996, and in revised form, February 11, 1997)
From the Department of Biochemistry and Molecular Biology, Marshall University School of Medicine, Huntington, West Virginia 25755
Retinoic acid (RA) induces differentiation of B16
mouse melanoma cells, which is accompanied by an increase in protein
kinase C (PKC
) as well as a selective enrichment of nuclear
PKC
. We report here that RA also increases AP-1 activity in these
cells. Transient transfection of B16 cells with luciferase reporter
gene constructs indicated that RA induced a
concentration-dependent increase in AP-1 activity. Acute
treatment (2 h) of B16 cells with phorbol dibutyrate (PDB) increased
AP-1 activity by 10-fold. RA treatment did not change the expression of
Jun family members; however, it decreased the expression of c-Fos. In
contrast acute PDB treatment induced c-Fos expression, while having
little effect on c-Jun. Five DNA-protein complexes were formed with
nuclear extracts from B16 cells and an oligonucleotide containing an
AP-1 consensus sequence. Several complexes were decreased in cells treated with RA. Conversely, certain complexes were increased in cells
acutely treated with PDB. The slowest migrating complexes were shown to
contain Fos family members. Down-regulation of PKC inhibited both the
acute PDB-induced and the RA-induced increase in AP-1 activity. The
selective PKC enzyme inhibitor, bisindolylmaleimide, reduced
PDB-stimulated AP-1 activity, but enhanced RA-induced AP-1 activity.
These results together with our previous studies suggest the intriguing
possibility that PKC protein, but not enzyme activity, may be required
for RA-induced AP-1 activity.
Retinoic acid (RA),1 a biologically active metabolite of vitamin A, has been shown to play an essential role in maintaining the differentiated phenotype in a variety of tissues (1, 2). This retinoid also induced differentiation in a number of tumor cell lines in culture (3-7). In B16 mouse melanoma cells, RA inhibits both anchorage-dependent and -independent growth, stimulates melanin production, and increases nerve growth factor receptors on the cell surface (8).
Several groups, including our own, have shown that the RA-induced
differentiation of certain tumor cells is accompanied by an increase in
PKC expression (5, 9-12). In B16 mouse melanoma cells RA induces a
5-8-fold increase in PKC protein (9, 10), which is accompanied by a
selective enrichment of nuclear-associated PKC
(13). Overexpression
of PKC
in these cells results in a more differentiated phenotype
(14), suggesting that this protein plays an important role in
RA-induced differentiation.
PKC is thought to regulate gene expression by altering the activity of
the AP-1 transcription complex (15). This complex is usually composed
of Jun family homodimers or Jun/Fos family heterodimers (15-18). There
is an interaction between retinoic acid receptors and the AP-1
transcription complex (19). This ligand (RA)-dependent
interaction results in a mutual loss of DNA binding activity to their
respective response elements (20). It has been suggested that this
interaction is the mechanism by which RA inhibits cell proliferation
(21). However, in RA-induced F9 teratocarcinoma differentiation, which
is accompanied by growth arrest, AP-1 activity/DNA binding is increased
(22). F9 cell differentiation is also correlated with an increase in
PKC protein. Overexpression of PKC
induces some of the
differentiation-dependent biochemical changes including
induction of c-jun expression (23).
In light of the RA-induced increase in PKC protein in B16 cells, we
determined whether there was any change in AP-1 activity. We report
here that RA treatment induces a 4-fold increase in AP-1 activity in
these cells. However, the mechanism appears to be quite different from
the increased AP-1 activity due to acute activation of PKC enzyme
activity.
B16 mouse melanoma cells were grown in a humidified atmosphere of 7% CO2, 95% air at 37 °C in Dulbecco's modified Eagle's medium. This medium contained 1 g/liter glucose and was supplemented with 10% heat-inactivated bovine calf serum (Sterile Systems, Logan, UT), 50 units/ml penicillin G, and 50 µg/ml streptomycin sulfate.
Retinoic AcidAll-trans-RA was obtained from Fluka Chemical Co. (New York). All experiments involving the use of RA were conducted in subdued light to prevent photo-oxidation of the retinoid. A concentrated stock solution of RA (10 mM) was prepared in ethanol. This stock solution was diluted to the desired final concentration in tissue culture medium and was sterile filtered before adding to the cells.
Northern BlottingRNA was isolated by a single-step method
as described previously (24). The RNA was then fractionated on 1%
agarose containing formaldehyde and transferred to Hybond N nylon
membranes (Amersham Corp.) by downward alkaline blotting (25). The
transferred RNA was cross-linked to the membrane by UV light. The
membrane was prehybridized for 1 h in 6 × SSC and 2% SDS.
32P-Labeled cDNA probes (1 × 106
dpm/ml) were then incubated with the membranes in fresh hybridization solution for 20 h. Blots were washed three times for 15 min each in 1 × SSC + 0.1% SDS, 0.5 × SSC + 0.1% SDS, and 0.2 × SSC + 0.1% SDS, respectively. The blots were exposed to Kodak XAR
film in cassettes at 70 °C for 2-5 days. All the cDNA probes
were labeled using the "prime-a-gene" labeling system from Promega
(Madison, WI) + 0.25 mCi of [
-32P]dCTP. The relative
amount of the different RNA species was quantitated by imaging the
autoradiogram with a Molecular Dynamics laser densitometer, making sure
that the signals were within the linear range of the instrument. The
data are expressed as the ratio of the specific mRNA to the
internal control, glyceraldehyde-3-phosphate dehydrogenase.
The following cDNA clones were used as probes in Northern blot analyses: pGEM-4 c-jun (rat) contains the complete 1.8-kb c-jun cDNA (26). Clone pSP65 c-fos (rat) contains the full-length 2.2-kb c-fos cDNA (27). Clone pHcGAP (glyceraldehyde-3-phosphate dehydrogenase) was purchased from the ATCC.
Western BlottingCells were seeded at 2.5 × 105/100-mm tissue culture dish. The following day cells were refed with Dulbecco's modified Eagle's medium with or without 10 µM RA. Cells were harvested at 24 and 48 h of incubation, by aspirating the growth medium, washing the cells twice with phosphate-buffered saline, and then preparing nuclear extracts by the method of Dignam et al. (28). Protein concentration was determined by using the BCA protein assay kit from Pierce. Proteins (50 µg) were fractionated using SDS-polyacrylamide gel electrophoresis with 10% separating and 5% stacking gels. Proteins were then transferred to Hybond-C membranes (Amersham Corp.) by using a semidry transfer cell. The membrane was incubated in blocking solution (Tris-buffered saline containing 0.2% Tween 20 and 5% nonfat dry milk, TBST) overnight. Blots were then incubated with 1 µg/ml polyclonal anti-c-Jun antibody (Ab-2, Oncogene Science, Mineola, NY), or with 1 µg/ml polyclonal anti-c-Fos antibody (Oncogene Science) for 1 h. They were then washed three times in TBST and incubated with 1:3000 dilution of secondary antibody (horseradish peroxidase-conjugated anti-rabbit IgG, Amersham Corp.) for 1 h. The membranes were washed three to five times in TBST, and signals were visualized by use of the ECL kit from Amersham Corp.
Reverse Transcription-PCRThe RNA PCR core kit provided by Perkin-Elmer was used for these assays. 1 µg of RNA from control or RA-treated B16 cells was converted to cDNA using Moloney murine leukemia virus reverse transcriptase with oligo(dT) (1.5 mM) as primers. The 30-µl reaction contained 1 × PCR buffer III, 3 mM MgCl2, 0.5 mM each of dATP, dTTP, dCTP, and dGTP, and 30 units of RNasin. RNA was denatured at 70 °C for 3 min and cooled on ice before adding the above reagents. Samples were incubated for 45 min at 42 °C and then 10 min at 80 °C.
PCR amplification of the cDNAs was performed using 50-µl
reactions containing 1 × PCR buffer II, 1.5 mM
MgCl2, 0.2 mM each of dATP, dCTP, dTTP, and
dGTP, plus 2.0 units of Taq polymerase, 1.0 mM
of each primer, and 10% of the cDNA synthesized in the RT
reaction. Primer sequences for c-jun were
(5-ACCCAGTTCTTGTGCCCCAA-3
), junB
(5
-AAACCCACCTTGGCGCTCAA-3
), and junD
(5
-CCGGATCTTGGGCTCCTCAA-3
) (sequence information kindly provided by
Dr. Steven Estus, Sanders-Brown Center on Aging, University of
Kentucky). All reactions were covered with a drop of mineral oil and
subjected to 17-25 PCR cycles. The typical reaction conditions were 1 min at 95 °C, 1 min at 55 °C, and 2 min at 72 °C. The
amplified cDNA products were separated by electrophoresis on 7.5%
polyacrylamide gels, stained with ethidium bromide, and visualized by
UV light. c-jun, junB, and junD
cDNAs were used as positive controls. Primers specific for
-actin were used to normalize the RNA in each RT
reaction.
An oligonucleotide
containing a consensus AP-1 sequence
(5-CGCATGAGTCAGACA-3
) was radiolabeled with
[
-32P]dCTP. Nuclear extracts from control and treated
cells (10 µg of protein) were incubated with 30,000 dpm of probe in a
buffer containing 10 mM Tris-HCl, pH 7.5, 1 mM
EDTA, 40 mM NaCl, 12% (v/v) glycerol, and 0.5 µg of
poly(dI-dC) in a total reaction volume of 25 µl. Where indicated,
1-2 µg of anti c-Jun or c-Fos antibodies (Santa Cruz Biotech, Santa
Cruz, CA) were added to the reaction mixture, which was then incubated
at room temperature for an additional 45 min. Samples were separated on
5% nondenaturing polyacrylamide gels
B16 cells were transfected with 4 µg of
pGL-2-AP-1 DNA or the pGL-2 vector alone + 1 µg of
SV40--galactosidase DNA to correct for transfection efficiency using
the calcium phosphate precipitation method (29). After an overnight
incubation, the transfection medium was removed, and the cells were
incubated with the various compounds as indicated under "Results."
Cells were harvested 48 h after transfection and assayed for
luciferase and
-galactosidase activity using kits from Promega. All
transfections were performed in triplicate dishes, and the experiments
were repeated three to five times.
Cells were lysed on ice in extraction
buffer (20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 0.5 mM EGTA, 1 mM dithiothreitol, 5% Trition X-100, 0.5 mM phenylmethylsulfonyl fluoride, and 10 µg/ml
aprotinin). Complete cell disruption was further ensured by three
consecutive 10-s sonications with a Tekmar sonic disrupter at power
setting 60. The total cell lysate was centrifuged at 12,000 × g for 15 min. The supernatant was loaded onto a
DEAE-cellulose anion exchange column (Cellex-D, Bio-Rad), previously
equilibrated with column buffer (20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 0.5 mM EGTA, 1 mM
dithiothreitol). The column was washed with 5 volumes of column buffer.
The PKC fraction was eluted with 2.5 column volumes of buffer
containing 100 mM NaCl and concentrated with a Centricon-10
microconcentrator (Amicon, Bedford, MA). Protein concentrations of
samples were determined by the Pierce BCA reagent. Samples were diluted
to equal protein concentrations (20 µg) and assayed with a
commercially available PKC assay system (Amersham Corp.) + 0.2 µCi of
[-32P] ATP in the presence and absence of
12-O-tetradecanoylphorbol-13-acetate and phosphatidylserine.
The system utilizes synthetic, PKC-specific, substrate peptides, which
become phosphorylated with the radiolabeled phosphate group from ATP.
At the end of the reaction, the radiolabeled peptide was separated from
the unincorporated 32P by the use of affinity paper for the
peptide. The degree of phosphorylation was determined by liquid
scintillation counting. Enzyme activity was calculated from counts/min
taking into account the specific activity of the radioisotope and
reaction time. Specific enzyme activity was obtained by subtracting the
counts/min obtained in the absence of the lipid mixture from that
obtained in the presence of the lipid mixture.
All experiments were repeated a minimum of three times. The data in most figures are from a representative experiment, which was qualitatively similar in the repeat experiments. Data from all transfection experiments are expressed as the mean ± S.E. of the mean of triplicate dishes of transfected cells for each treatment group. Since transfection experiments can have a higher degree of variability, these experiments were repeated a minimum of four to five times with similar qualitative results.
Phorbol ester-activated PKC
stimulates AP-1 activity (15). In light of the 6-8-fold increase of
PKC protein in RA-treated B16 melanoma cells (10) and a selective
enrichment of nuclear-associated PKC
(13), we determined whether RA
also increased AP-1 activity. B16 melanoma cells were transiently
transfected with a pGL-2 plasmid containing four tandem AP-1 consensus
sequences inserted 5
to an SV-40 promoter driving the expression of
luciferase. Transfected cells were treated for 24 h with or
without various concentrations of all-trans-retinoic acid.
As a positive control, one group of transfected cells was treated with
1 µM PDB for the last 2 h of incubation. At the end
of the incubation period (48 h from the start of transfection), all
cells were harvested and assayed for luciferase and
-galactosidase
activity. These experiments (Fig. 1) revealed that B16
cells have endogenous AP-1 activity (pGL-2 without AP-1 sequences gave
only background levels of luciferase activity) and that this activity
is stimulated 9-fold by PDB. RA increased AP-1 activity 2-fold at the
lowest concentration (10 nM), and the stimulation increased
to 4-fold at the highest RA concentration (10 µM).
Effect of RA and PDB on Expression of Fos and Jun Family Members
The AP-1 transcription complex is most commonly composed
of c-Jun homodimers or c-Jun/c-Fos heterodimers. We examined the possibility that the RA and/or PDB-induced increase in AP-1 activity could be due to an increase in the expression of one of these transcription factors. The amount of c-jun and
c-fos mRNA in control, PDB-treated, or RA-treated B16
cells was examined by Northern blotting. Two messages (3.2 and 2.6 kb)
for c-jun were found in B16 cells (Fig.
2A). The 2.6-kb mRNA was the predominant
form in these cells. c-jun mRNA was not consistently
altered by RA treatment. The small increases depicted in this blot were
not always seen in replicate experiments. In contrast, a 1-h treatment with PDB increased the amount of c-jun mRNA by 2-fold.
B16 cells also express a 2.1-kb c-fos mRNA (Fig.
2B). Treatment of the cells with RA led to a consistent
decrease in this c-fos message, while a 1-h treatment with
PDB induced a 5-fold increase in the amount of c-fos
mRNA. We were not able to detect junB or junD
mRNA by Northern blotting. Semiquantitative reverse
transcription-PCR was employed to examine the expression of these
jun family members. We found that these genes were expressed
in B16 cells, but after ensuring a linear signal (by varying the number
of PCR cycles and correcting by -actin levels), we found no
change in junB or junD mRNA levels (data not
shown).
To determine if the changes in mRNA were reflected in the amount of
c-Jun or c-Fos protein, we treated B16 cells for different times with
RA and measured the amount of the appropriate protein by Western
blotting. B16 cells express a 39-kDa immunoreactive c-Jun protein (Fig.
3A). There was no consistent increase in the amount of this protein in RA-treated cells compared with untreated cells. The antibody used to detect c-Fos protein also recognizes Fos B
as well as fra-1 and fra-2 (Fig. 3B, NIH 3T3 cell extract used as a control). In control cells no signal was detected at 6 h, but these cells had increasing amounts of c-Fos protein at 24 and
48 h of incubation. The expression of other Fos family members was
barely detectable in nuclear extracts from B16 cells. Treatment of
these cells for 48 h with RA markedly decreased (60% of control)
the amount of c-Fos protein compared with control cells at the same
time point (Fig. 3B).
Since PDB induced a large increase in c-fos mRNA, we
examined the time course for this induction at both the RNA and protein level. A 1-h PDB treatment increased c-fos mRNA by
4-fold (Fig. 4A). This increase was transient
and returned to unstimulated levels by 12 h despite the continued
presence of PDB. PDB also increased the level of c-Fos protein within
2 h of treatment (Fig. 4B). Similar to the increase in
c-fos mRNA, the increase in protein was also transient
and returned to unstimulated levels by 12 h. Fos B and fra-1
proteins were also increased by PDB, but the rate of increase was
slower than for c-Fos (Fig. 4B).
Effect of RA and PDB on AP-1 Binding Activity
Since RA did
not increase the amount of any of the AP-1 family members, we
determined whether an increase in binding activity might account for
the RA-induced increase in AP-1 transcriptional activity. Using
electrophoretic mobility shift assays with a radiolabeled 15-base pair
oligonucleotide containing one AP-1 consensus site, five protein-DNA
complexes were observed (Fig. 5A). Complex 5 was absent when nuclear extracts from 24- or 48-h RA-treated cells were
analyzed. Nuclear extracts from 48-h RA-treated cells were also missing
complex 1, while the binding activity of complexes 2 and 4 were reduced
compared with control cells (Fig. 5A). In contrast, nuclear
extracts from B16 cells treated for 2 h with PDB had markedly
increased binding activity of complex 2. As the time of PDB treatment
was increased, binding activity of complexes 1 and 2 decreased, but the
activity of complexes 4 and 5 increased dramatically. Incubation of
nuclear extracts with antibodies to c-Jun did not change the intensity
of any bands, nor were any bands supershifted. In contrast, incubation
of the nuclear extracts with c-Fos antiserum decreased the binding
activity of complexes 1 and 2 and also induced the formation of a
supershifted complex. Competition experiments with nonradioactive wild
type and mutant AP-1 oligonucleotides indicated the following
sensitivity: complex 1 > complex 4 > complex 2 = complex 3 > complex 5. All five complexes were still present at a
50-fold excess of the mutant oligonucleotide (Fig. 5B).
Role of PKC in RA-induced AP-1 Activity
RA-induced
differentiation of B16 cells is accompanied by a 6-8-fold increase in
PKC mRNA and protein. PDB, via its activation of PKC, is known
to increase AP-1 activity. Therefore we investigated whether PKC was
required for the RA-induced increase in AP-1 activity in B16 melanoma
cells. We down-regulated PKC protein in cells transfected with the
AP-1-luciferase reporter gene by chronic PDB treatment and measured
their ability to respond to RA. We also treated the transfected cells
with the selective PKC inhibitor bisindolylmaleimide and measured the
ability of RA or PDB to increase AP-1 activity. Fig.
6A shows that down-regulation of PKC
inhibited both the acute PDB-induced (lane 2 versus 5) and
the RA-induced (lane 3 versus 6) increase in AP-1 activity.
Western blot analysis (Fig. 6B) shows that RA induced
PKC
, while chronic PDB treatment depleted the cells of PKC
. RA + PDB-treated cells have PKC
levels slightly lower than control cells,
but higher than cells treated with PDB for 24 h. In separate
experiments we tested the effect of inhibition of PKC enzyme activity
on the PDB and RA-induction of AP-1 transcriptional activity. We found
that PKC enzyme activity from B16 cells was reduced by 90% at 0.1 µM bisindolylmaleimide and was not detectable at 1.0 µM concentration of this inhibitor (Fig.
7A). When this inhibitor was added to the
transfected cells, it reduced the PDB-induced AP-1 activity from
11-fold down to 4-fold. In contrast, the inhibitor enhanced RA-induced
AP-1 activity from 5-fold to 8-fold. To determine if changes in the
level of PKC
might explain these unanticipated results, Western blot
analysis was performed on cells treated/untreated with 10 µM RA, 2 µM bisindolylmaleimide, or a
combination of these two compounds. We found an increase in PKC
protein in inhibitor-treated cells compared with the controls. PKC
protein was also increased in RA + inhibitor-treated cells compared
with RA-treated cells (Fig. 7C).
We have demonstrated that RA increases AP-1 transcriptional activity in a dose-dependent manner. This is in marked contrast to several studies in which RA inhibited AP-1 transcriptional activity (19, 20). RA-induced F9 teratocarcinoma differentiation, however, is also accompanied by an increase in c-jun expression and enhanced AP-1 binding activity (22). Furthermore, ectopic expression of c-jun leads to differentiation of P19 embryonal carcinoma cells in the absence of RA (23), suggesting a critical role for c-Jun in RA-induced differentiation.
We examined the possibility that, similar to F9 teratocarcinoma cells, RA increases the expression of one of the members of the AP-1 transcription complex. RA did not increase the expression of any fos or jun family member, but instead decreased the expression of c-fos mRNA and c-Fos, Fos B, and fra-1 proteins. Busam et al. (30) previously reported that RA decreased mitogen-induction of c-fos mRNA. They also found that the induction of c-jun mRNA was suppressed, but required higher concentrations of RA and a longer period of incubation. We found that the mitogen, PDB, transiently increased c-Fos family member protein expression in B16 cells. Interestingly, Fos B and fra-1 increased more slowly than c-Fos. Delayed expression of fra-1 and fra-2 with serum stimulation has previously been reported (31). A possible explanation may lie in the observation that c-Fos can transactivate the fra-1 and fra-2 genes (32). These data suggest that the RA-induced increase in AP-1 transcriptional activity may not involve a c-Jun/c-Fos heterodimer.
Since an increase in members of the AP-1 transcription complex could
not explain the RA-induced increase in AP-1 transcriptional activity,
we examined the possibility that RA altered AP-1 DNA binding activity.
Using a consensus oligonucleotide from the AP-1 site in the collagenase
gene and nuclear proteins from untreated B16 cells, we observed five
protein-DNA complexes. Instead of enhancing binding, RA inhibited the
appearance of complex 5 and with longer times of incubation (48 h) also
inhibited the appearance of complex 1. Also, the intensity of complexes
2 and 4 was reduced by a 48-h treatment of the B16 cells with RA. Short
term (2 h) treatment of cells with PDB increased the intensity of
complex 2, but with longer times of treatment this change was reversed, and the intensity of complexes 4 and 5 dramatically increased. Since a
Fos antiserum diminished the intensity of complexes 1 and 2 and caused
a "supershifted" complex, we conclude that these complexes contain
members of the Fos family. A variety of Jun antisera failed to diminish
the appearance or to "supershift" any complex. Some of these same
antisera successfully recognized c-Jun on Western blots. Since the
electrophoretic mobility shift assay is conducted under nondenaturing
conditions, one explanation for these results is that the epitope
recognized by the antibodies is unavailable under the electrophoretic
mobility shift assay conditions. Alternatively, c-Jun may not be
involved in AP-1 complexes using our assay conditions (B16 melanoma
nuclear extracts, specific AP-1 oligonucleotide). The acute PDB-induced
increase in complex 2 and its decrease in RA-treated cells probably
reflects the opposite effect of these agents on the expression of
c-Fos. Since we have shown that chronic treatment with PDB depletes B16
cells of PKC, the major isotype expressed by these cells (33), the
decrease in complex 2 and the increase in complexes 4 and 5 may reflect a change in the expression of AP-1-associated transcription factors regulated through the PKC pathway. Overall, these results suggest that
the RA-induced increase in AP-1 transcriptional activity cannot be
explained by an increase in binding activity.
Finally, we examined the role of PKC in the RA-induced increase in AP-1
transcriptional activity. We found that down-regulation of PKC, through
chronic PDB treatment, inhibited both acute PDB and RA-induced increase
in AP-1 transcriptional activity. However, the PKC-specific enzyme
inhibitor, bisindolylmaleimide, enhanced RA-induced AP-1
transcriptional activity, while inhibiting acute PDB stimulation of
AP-1 transcriptional activity by 60%. These data lead to a tentative
conclusion that PKC protein, but not PKC enzyme activity, is required
for the RA induction of AP-1 activity. It has been reported that PKC
stimulates phospholipase D activity through a nonenzymatic mechanism
(34). Also PKC binds to proteins other than substrates (35, 36).
Inhibition of PKC enzyme activity also increased the amount of PKC
protein (Fig. 7C). A likely explanation for this result is
the enhanced proteolysis of activated PKC (37), which would be
diminished in bisindolylmaleimide-treated cells. The finding that
inhibition of PKC enzyme activity enhanced RA-stimulated AP-1
transcriptional activity correlates with other data presented in this
study. It reinforces the conclusion that activation of PKC enzyme
activity (via PDB) increases AP-1 activity by a different pathway than that induced by RA. Our data also suggest that the two pathways are
antagonistic to each other. This might provide a molecular explanation
for the antagonistic action of phorbol esters and RA on B16 melanoma
growth and differentiation (10, 33). It is important to note that,
while both agents increase AP-1 transcriptional activity, the effect of
PDB is transient, while RA has a prolonged effect (elevated through at
least 36 h of treatment; data not shown). Sustained elevation of
AP-1 activity might be required for activation of pathways leading to
differentiation in these melanoma cells. Current investigation is
focused on the mechanism by which RA increases AP-1 transcriptional
activity and the consequence of this activity for RA-induced inhibition
of cell proliferation and differentiation.
We thank Dr. Tom Curran for supplying us with the c-jun and c-fos plasmids and Dr. Steven Estus, Sanders-Brown Center on Aging, University of Kentucky, for supplying us with sequence information for the primers used in the reverse transcription-PCR detection of Jun family members.