Molecular Physiology and Genetics Section, Laboratory of Cellular and Molecular Biology, Gerontology Research Center, Baltimore, Maryland 21224
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ABSTRACT |
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We examined epidermal growth factor (EGF)- and
epinephrine-stimulated mitogen-activated protein kinase kinase (MEK) 1 and MEK2 activities, DNA polymerase activity, and EGF-stimulated E2F DNA binding activity in primary cultured hepatocytes from 6- and
24-mo-old rats. MEK stimulation by either EGF or epinephrine was not
altered with aging. However, stimulation of DNA polymerase
activity
by these agents was 70% and 50% lower, respectively, in cells of aged
compared with cells of young rats, consistent with a lesser increase in
[3H]thymidine
incorporation. EGF-stimulated E2F (a transcription factor that
regulates expression of the DNA polymerase
gene) binding to DNA was
reduced with age. PD-098059, a specific inhibitor of MEK, inhibited
EGF-stimulated MEK1 and MEK2 activities in hepatocytes from 6- and
24-mo-old rats. Although PD-098059 inhibited EGF-stimulated DNA
synthesis in hepatocytes from 6-mo-old rats, it had no effect in
24-mo-old rats. Thus the age-related impairment appears to occur before
E2F activation, and signal transduction sequences other than the
mitogen-activated protein kinase pathway may be involved in stimulated
DNA synthesis in hepatocytes from old rats.
aging; epidermal growth factor; polymerase
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INTRODUCTION |
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ALTERATIONS IN THE regulation of DNA synthesis and cell division represent some of the most important functional manifestations of aging (6). We and others have recently developed an in vivo model of cellular aging, primary cultures of rat hepatocytes, which exhibit impaired stimulation of DNA synthesis with increasing donor age (20, 21, 31). Epidermal growth factor (EGF), epinephrine, and isoproterenol are all less effective in stimulating DNA synthesis in cultures obtained from aged rats than in cells from young animals (20, 21, 31). Because growth factors and catecholamines exert their effects through widely different signal transduction pathways (e.g., tyrosine kinases vs. G protein-linked receptors), it seems likely that defects in stimulation of DNA synthesis occur at a very fundamental level.
Many receptor tyrosine kinases, cytokine receptors, and seven-membrane spanning receptors that couple to heterotrimeric G proteins have been shown to activate mitogen-activated protein kinases (MAPKs), which has one subfamily also known as extracellular signal-regulated kinases (ERKs) (3). MAPK activation is associated with both cell proliferation and differentiation (35). A common pathway leading from cell surface receptors to MAPKs involves the small GTP-binding protein Ras, which binds to the c-Raf protein kinase and contributes to its activation (38). Raf has been shown to phosphorylate and activate MAPK kinase, also known as MEK (MAPK or ERK kinase) (10), which in turn phosphorylates and activates MAPKs (18). MAPKs translocate to the nucleus on activation. Many transcription factors contain potential MAPK phosphorylation sites, and MAPK clearly plays a role in activation of some transcription factors (19).
The transition of quiescent cells to active proliferation is
characterized by a temporal program of gene expression (29). Genes that
display induced expression during this program can be classified into
two groups: early- and late-response genes. The expression of
early-response genes does not require protein synthesis and is
frequently superinduced by cycloheximide. Late-response genes generally
do require prior protein synthesis, as cycloheximide can prevent their
expression. Many genes encoding enzymes and proteins involved in either
nucleotide metabolism or DNA replication are late-response genes. The
enzyme activities or protein levels of dihydrofolate reductase,
thymidine kinase, thymidylate synthase, DNA polymerase , and others
increase shortly before, or simultaneously with, the onset of DNA
synthesis (39). One thing these late-response genes have in common is
their dependence on the transcription factor E2F (27).
Previous studies have determined that EGF receptors are unaltered with
age (20, 31) and suggested that EGF activation of MAPK is reduced,
possibly due to elevation in the level of MAPK phosphatase (25).
However, it is not clear whether these changes are sufficient to fully
explain the age-related impairments in stimulated DNA synthesis by both
catecholamines and growth factors. It thus became important to examine
other events that might be responsible for the age changes. The present
study, therefore, compares EGF- and epinephrine-stimulated MEK1 and
MEK2 activities, DNA polymerase activity, and E2F DNA binding
activity in hepatocytes of young and old rats.
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METHODS |
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Animals. Male Wistar rats, ages 6 and 24 mo, were obtained from the Gerontology Research Center (National Institute on Aging) colony. These rats have a mean life span (50% mortality) of ~23 mo. Rats were maintained on 12:12 h light-dark cycles in a controlled environment and fed National Institutes of Health Purina Chow ad libitum.
Chemicals.
Collagenase (type 2) was purchased from Worthington Biochemical
(Freehold, NJ). Collagen (type 1), dexamethasone, aprotinin, trypsin
inhibitor, BSA, ()-epinephrine, 2'-deoxyadenosine
5'-triphosphate (dATP), 2'-deoxycytidine
5'-triphosphate (dCTP), 2'-deoxyguanosine 5'-triphosphate (dGTP), deoxythymidine 5'-triphosphate
(dTTP), activated calf thymus DNA, phenylmethylsulfonyl fluoride
(PMSF), EGTA, EDTA, and myelin basic protein (MBP) were obtained from Sigma Chemical (St. Louis, MO). EGF was obtained from GIBCO BRL (Gaithersburg, MD). Hanks' balanced salt solution and Williams medium
E were obtained from Quality Biological (Gaithersburg, MD).
[Methyl-3H]thymidine
(85 Ci/mmol) and
[methyl,1',2'-3H]thymidine
5'-triphosphate (117 Ci/mmol) were obtained from Amersham (Arlington Heights, IL).
[
-32P]ATP (4,500 Ci/mmol) was obtained from ICN Pharmaceuticals (Irvine, CA). Anti-MEK1
and anti-MEK2 monoclonal antibodies were obtained from Transduction
Laboratories (Lexington, KY). E2F consensus oligonucleotide and
anti-E2F1 monoclonal antibody were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA). Glutathione
S-transferase fusion
[K71A]ERK1 (GST-ERK1) agarose conjugate was obtained from Upstate Biotechnology (Lake Placid, NY). Pansorbin cells were obtained
from Calbiochem-Novabiochem (San Diego, CA). PD-098059 was a gift of
Dr. Alan R. Saltiel of Parke-Davis Pharmaceutical Research Division.
All other chemicals used were the highest grade commercially available.
Primary hepatocyte cultures.
Rat hepatocytes were isolated by the collagenase perfusion methods as
described before (21). The isolated cells were suspended at 2.5 × 105 cells/ml in Williams medium E
containing 0.1 µg/ml aprotinin and
109 M dexamethasone
supplemented with 5% FCS and were plated into 22-mm-diameter wells of
multiwell culture plates and 10-cm-diameter dishes, which had been
coated with rat type 1 collagen. Cells were cultured at 37°C under
5% CO2 in air for 3 h to allow
attachment to the dishes; the medium was then replaced with serum-free
Williams medium E containing 0.1 µg/ml of aprotinin and
10
9 M dexamethasone. Cell
viability was determined by trypan blue dye exclusion before plating.
Assessment of DNA synthesis.
DNA synthesis was assessed by measuring incorporation of
[3H]thymidine into DNA
as described before (21). Either EGF (100 ng/ml) or epinephrine
(104 M) was added 20 h
after cell inoculation, and then, at appropriate times,
[3H]thymidine (10 µCi/ml, 85 Ci/mmol) was added. After 2 h, the cells were washed twice
with PBS and immersed in 1 ml of 10% TCA. The hepatocytes were
solubilized by incubation at 37°C for 30 min in 0.5 ml of 1 N NaOH,
and 100% TCA was added to the solution to a final concentration of
10%. The precipitate was washed twice with 10% TCA and hydrolyzed by
heating at 90°C for 15 min in 0.5 ml of 10% TCA. Radioactivity in
the hot TCA-soluble fraction was measured by liquid scintillation
counting. Protein was measured by the method of Lowry et al. (26) using
BSA as a standard.
Measurement of MEK1 and MEK2 activity.
Cells were washed once with PBS and lysed in ice-cold lysis buffer (10 mM Tris, pH 7.4, 1% Triton X-100, 0.5% Nonidet P-40, 150 mM NaCl, 20 mM sodium fluoride, 0.2 mM sodium orthovanadate, 1 mM EDTA, 1 mM EGTA,
and 0.2 mM PMSF) and centrifuged at 12,000 g for 30 min at 4°C. After
normalization for protein, as determined by Bradford analysis (20), the
lysates were incubated with either anti-MEK1 or anti-MEK2 monoclonal
antibodies, followed by addition of 50 µl of Pansorbin. The
immunocomplexes were washed twice in the immunoprecipitation buffer (10 mM Tris, pH 7.4, 1% Triton X-100, 0.5% Nonidet P-40, 150 mM NaCl, 0.2 mM sodium orthovanadate, 1 mM EDTA, 1 mM EGTA, and 0.2 mM PMSF),
followed by two washes in kinase buffer (50 mM
Tris · HCl, pH 8.0, 3 mM magnesium acetate, 1 mM
EGTA, 5 mM dithiothreitol, and 0.1 mg/ml ovalbumin). Forty microliters
of the kinase buffer containing 50 µM ATP, 5 µCi
[-32P]ATP, and
either 5 µg of GST-ERK1 (1) or 40 µg MBP (32) as a
substrate. As previously reported (32), MBP was phosphorylated by MEK,
and this phosphorylation correlated with MEK activity, although
phosphorylation of MBP by MEK was lower in sensitivity than that of
GST-ERK1. Therefore, to assay for MEK activity, both GST-ERK1 and MBP
were used as substrates. The reactions were carried out at 30°C for
20 min; reactions were terminated by the addition of 15 µl of
5× concentrated Laemmli sample buffer (24) and then boiled for 5 min. The samples were centrifuged at 12,000 g for 3 min, and the soluble fractions were electrophoresed. The gels were
dried and exposed to film. Quantitation of exposed films was carried
out by using a personal densitometer (Molecular Dynamics, Sunnyvale,
CA).
Measurement of DNA polymerase activity.
Activities of DNA polymerase
in the cultured hepatocytes were
assayed by the method of Friedman et al. (16) with some modifications.
Cells were washed once with PBS and lysed in 500 µl of ice-cold Tris
buffer (pH 7.6) containing 100 µM EDTA, 200 µM dithiothreitol, 500 µM KCl, 10% glycerol, and 0.5% Triton X-100. The lysates were
subjected to a brief ultrasonication and centrifuged at 15,000 g for 30 min at 4°C, followed by
30,000 g for 90 min. The resulting
supernatants were used for DNA polymerase assays. The 500-µl reaction
mixtures consisted of 50 mM Tris · HCl (pH 7.4), 1 mM
dithiothreitol, 0.8 mM MgCl2, 50 µM dCTP, dGTP, and dATP, and
[3H]dTTP [~60
counts · min
1
(cpm) · pmol
1],
15% glycerol, 400 µg/ml BSA, 3 mM KCl, and 80 µg/ml activated calf
thymus DNA with and without 30 µg/ml aphidicolin. Then, 100 µl of
cell extract adjusted to contain 100 µg of protein were added, and
the mixtures were incubated for 60 min at 37°C. The reaction
mixtures were collected on DEAE-cellulose filters and washed with 5%
TCA. The radioactivities were determined by liquid scintillation
counting. DNA polymerase activity was calculated by subtracting the
values with aphidicolin from those without aphidicolin.
Gel retardation analysis.
Nuclear extracts were prepared by the method of Dignam et al. (12).
Oligonucleotide containing a consensus E2F-1 binding sequence
(5'-ATTTAAGTTTCGCGCCCTTTCTGAA-3') was
32P labeled with
[-32P]ATP, using
polynucleotide kinase (50,000 cpm/ng). Binding reaction mixtures (50 µl) containing 0.5 ng of DNA probe, 20 µg of nuclear extract, 0.33 M urea, 0.10 M NaCl, 0.33% Nonidet P-40, 25 mM HEPES (pH 7.9), 10 mM
DTT, 10% glycerol, 5 µg of BSA, and 3 µg of poly(dI-dC) were
incubated at room temperature for 30 min. For DNA binding competitions,
a 100× molar excess of unlabeled E2F-1 consensus oligonucleotide
was added to the binding reaction. The control nuclear extract was
provided by Santa Cruz Biotechnology. DNA-protein complexes were
resolved by electrophoresis through 4% polyacrylamide gels containing
50 mM Tris, 0.38 M glycine, and 2 mM EDTA. The gels were subsequently
dried and autoradiographed.
Western blot analysis. Cells were washed once with PBS and lysed in 500 µl of Laemmli sample buffer (24). The lysates were normalized to equivalent total cellular protein levels, as determined by Bradford analysis (4), and electrophoresed on 10% polyacrylamide gels. Proteins were transferred to nitrocellulose paper as described by Towbin et al. (36) and immunoblotted with antibody. Immune complexes were visualized using the enhanced chemiluminescence procedure (Amersham). Quantitation of exposed films was carried out by using a personal densitometer (Molecular Dynamics).
Statistical analysis. All values are expressed as means ± SE. Statistical analysis was performed by one-way ANOVA. Differences between individual age or treatment groups were evaluated using the unpaired two-tailed Student's t-test.
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RESULTS |
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Effect of aging on EGF- or epinephrine-stimulated DNA synthesis. Hepatocyte DNA synthesis in response to EGF was assessed using a 2-h pulse of [3H]thymidine. We demonstrated previously that [3H]thymidine incorporation in cultured hepatocytes from rats of various ages increased 12 h after addition of EGF, reached a peak time at 48 h, and then gradually decreased (20). Similar results were obtained with stimulation by epinephrine (21). Therefore, we examined [3H]thymidine incorporation 48 h after addition of EGF or epinephrine to primary cultures from 6- and 24-mo-old rats to be certain that DNA synthesis was indeed reduced in the aged hepatocytes under examination. As shown in Fig. 1, both EGF (A)- and epinephrine (B)-stimulated DNA syntheses in hepatocytes from 6- and 24-mo-old rats were significantly (P < 0.05) increased over unstimulated control levels. The levels of EGF- and epinephrine-stimulated DNA syntheses in hepatocytes from 24-mo-old rats were both significantly (P < 0.05) lower than that from 6-mo-old rats, although the control levels were not significantly different. The increases in DNA synthesis 48 h after addition of EGF or epinephrine in 24-mo-old rats were reduced about 70 and 50% compared with 6-mo-old rats, respectively. Similar results are obtained when the data are expressed per milligram protein rather than per cell. Hence, all subsequent results are presented in the latter format.
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Effect of aging on EGF- or epinephrine-stimulated MEK1 and MEK2 activities. To determine whether EGF- and epinephrine-stimulated MEK1 and MEK2 activities are reduced with age, we examined EGF- and epinephrine-stimulated MEK1 and MEK2 activities in hepatocytes from 6- and 24-mo-old rats. Neither MEK1 nor MEK2 protein levels changed in response to EGF treatment or differed between young and old cells (Fig. 2A). Optical density (OD) values (means ± SE from 3 experiments) were as follows for MEK1: young 0 min = 1,110 ± 69, young 5 min = 1,042 ± 96, young 30 min = 1,164 ± 59, old 0 min = 1,108 ± 69, old 5 min = 855 ± 99, and old 30 min = 1,097 ± 90. For MEK2, OD values were as follows: young 0 min = 2,145 ± 146, young 5 min = 2,302 ± 200, young 30 min= 2,206 ± 152, old 0 min = 2,072 ± 160, old 5 min = 1,945 ± 212, and old 30 min = 2,325 ± 196. There are no statistically significant effects of age on EGF levels with either enzyme. Similarly, EGF and epinephrine significantly stimulated (P < 0.01) both MEK1 and MEK2 activities in primary cultured hepatocytes from 6- and 24-mo-old rats (Figs. 2B and 3). As shown in Figs. 2B and 3, both MEK1 and MEK2 activities reached a maximum at 5 min after EGF stimulation and at 10 min after epinephrine stimulation. EGF- and epinephrine-stimulated MEK1 and MEK2 activities were not significantly altered during aging. Furthermore, the addition of EGF to primary cultured hepatocytes from 6- and 24-mo-old rats stimulated both MEK1 and MEK2 activities in a concentration-dependent manner.
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Effect of aging on EGF- or epinephrine-stimulated DNA polymerase
activities.
To determine whether the incorporation of
[3H]thymidine reflects
the activity of DNA polymerase, we examined EGF- or
epinephrine-stimulated DNA polymerase
activities in primary
cultured hepatocytes from 6- and 24-mo-old rats. As demonstrated in
Table 1, EGF- and
epinephrine-stimulated DNA polymerase
activities in hepatocytes
from 6- and 24-mo-old rats were both significantly increased over
unstimulated control levels. The levels of EGF- and
epinephrine-stimulated DNA polymerase
activity in hepatocytes from
24-mo-old rats were both significantly lower than those in 6-mo-old
rats, although the control levels were not significantly different. The
increases in DNA polymerase
activity 48 h after addition of EGF or
epinephrine in 24-mo-old rats were reduced about 70% and 50%,
respectively, compared with 6-mo-old rats.
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Effect of aging on EGF-stimulated E2F DNA binding activity. To determine whether changes in E2F DNA binding activity during the course of EGF stimulation might contribute to age differences in DNA synthesis, we examined E2F1 protein expression by Western blot analysis and E2F DNA binding activity by gel retardation analysis in primary cultured hepatocytes from 6- and 24-mo-old rats 0 and 48 h after the addition of EGF. EGF significantly increased (P < 0.01) both E2F DNA binding activity and E2F1 protein expression in hepatocytes from both 6- and 24-mo-old rats (Fig. 4A). Although EGF-stimulated E2F1 protein expression did not differ between young and old cells, the EGF-stimulated E2F DNA binding activity in hepatocytes from 24-mo-old rats was significantly (P < 0.05) lower than that seen in 6-mo-old rats (Fig. 4B). The ratio of EGF-stimulated activity to basal E2F binding activity was 1.24 ± 0.05% vs. 1.81 ± 0.13% in old and young animals, respectively (P < 0.05). The specificity of the DNA binding activity was assessed in two ways. First, the mobility of the E2F DNA binding activity in hepatocyte extracts is coincident with that seen in k-ras transformed kidney cell nuclear extracts (Fig. 4B, lanes marked C) following treatment with phorbol ester. Second, addition of unlabeled E2F1 consensus oligonucleotide to the binding reactions of 48-h-treated young hepatocyte extracts or control nuclear extracts successfully competed for DNA binding.
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Effect of PD-098059 on EGF-stimulated MEK1 and MEK2 activities and DNA synthesis in hepatocytes from 6- and 24-mo-old rats. To determine whether the MAPK pathway is the only pathway of EGF-stimulated DNA synthesis, we employed the recently identified specific inhibitor of MEK activity, PD-098059. This reagent is noncompetitive for ATP and does not affect the enzyme activities of over 30 tyrosine and serine/threonine kinases examined, including the highly related JUN kinase, JNKK (24, 25). PD-098059 inhibited both EGF-stimulated MEK1 and EGF-stimulated MEK2 activities in hepatocytes from 6- and 24-mo-old rats (Fig. 5A). Surprisingly, however, although PD-098059 inhibited EGF-stimulated DNA synthesis in hepatocytes from 6-mo-old rats, it had no effect on that from 24-mo-old rats (Fig. 5B).
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DISCUSSION |
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Present studies have demonstrated that neither MEK1 stimulation nor MEK2 stimulation by either EGF or epinephrine appears to be altered with aging, although EGF- and epinephrine-stimulated DNA syntheses decrease. These findings suggest that no age change exists from receptor binding (20, 31) to MEK activation. It has been reported that, on the basis of Northern blots, rat and murine MEK2 are highly expressed in liver, a tissue in which MEK1 is very low (17, 40). In this study, protein levels of MEK1 also appear lower than those of MEK2, but such differences could be secondary to differences in the respective affinities of the MEK1 and MEK2 antibodies. More importantly, however, activities of MEK1 and MEK2 were similar following EGF or epinephrine stimulation in primary cultured hepatocytes from 6- and 24-mo-old rats.
On the other hand, the increase in DNA polymerase activity
following stimulation by either EGF or epinephrine was reduced during
aging by 70% and 50%, respectively, consistent with the lower
stimulation of
[3H]thymidine
incorporation in cells of aged rats. Incorporation of
[3H]thymidine reflects
the activities of both thymidine kinase and DNA polymerases. Five types
of DNA polymerases have been defined in mammalian cells: DNA polymerase
,
,
,
, and
(17, 35). DNA polymerase
is the main
enzyme responsible for semiconservative DNA replication (13), whereas
the
,
, and
enzymes are thought to also play roles in DNA
repair (15). The
,
, and
polymerases are all sensitive to
aphidicolin (35). Only DNA polymerase
has significant activity on
nuclease-activated calf thymus DNA templates (8). Thus we determined
the fraction of polymerase activity that was specifically catalyzed by
DNA polymerase
by using a specific inhibitor and template.
Pendergrass et al. (30) showed that the levels of DNA polymerase
activity per cell in senescent human diploid fibroblast cells are
similar to that in young cells, although DNA polymerase
activity
per milligram protein is reduced in aged cells compared with young
cells. The observation that DNA polymerase
activity is
reduced in aged cells when normalized to protein content reflects
increased cell size in fibroblast cultures, a phenomenon that has been
previously documented (33). In contrast, the cell size and protein
content in primary cultured hepatocytes from 6- and 24-mo-old rats were similar (data not shown). These findings suggest that the levels of DNA
polymerase
activity per cell are also reduced in aged rat
hepatocytes.
Stimulation of DNA polymerase activity by EGF or epinephrine was
decreased with age, consistent with E2F (a transcription factor that
regulates expression of the DNA polymerase
gene) DNA binding
activity being reduced with age. However, levels of E2F1 protein
expression were comparable in mature and aged hepatocytes. Thus
defective molecules with reduced ability to be activated for DNA
binding may exist in aged cells. Alternatively, E2F1 may form complexes
with various molecules to a different extent in young and aged cells,
which might also result in impaired polymerase activation. It has been reported that the levels of E2F
mRNA fluctuate throughout the cell cycle. Little E2F mRNA is present in
quiescent 3T3 cells (34) or in resting T cells (22). However, the
levels of E2F mRNA increase at the
G1/S-phase boundary after serum
stimulation of 3T3 cells (34) and mitogen stimulation of resting human
T cells (22). E2F is also regulated by association with different cellular proteins in different stages of the cell cycle. In the G1 phase, E2F binds to the Rb
protein (5); this association continues through the S phase (33). Rb
undergoes cell cycle-dependent phosphorylation, with
underphosphorylated Rb present in
G0 and G1 phase cells and phosphorylated
Rb present in S and G2/M phase cells (9). E2F also forms a complex with p107, the kinase cdk2, and
cyclin E (in mid- to late-G1) or
cyclin A (in S phase) (11). All complexes appear to dissociate in
G2/M phase, resulting in free E2F.
Present findings suggest that the age-related impairment in stimulated
DNA synthesis may occur before E2F activation.
PD-098059, a specific inhibitor of MEK, inhibited both EGF-stimulated
MEK1 and EGF-stimulated MEK2 activities in hepatocytes from 6- and
24-mo-old rats. Although PD-098059 inhibited EGF-stimulated DNA
synthesis in hepatocytes from 6-mo-old rats, it had no effect on that
from 24-mo-old rats. These findings suggest that signal transduction
sequences other than the MAPK pathway are involved with EGF-stimulated
DNA synthesis in hepatocytes from 24-mo-old rats. Figure
6 shows signal transduction in stimulated
DNA synthesis. In fibroblasts, the increase in p21 expression with age
(28) inhibits Rb protein phosphorylation and decreases the level of free E2F. This decreases DNA polymerase transcription and DNA synthesis. In hepatocytes, although the expression of p21 decreases with age (23), E2F DNA binding activity also decreases with age.
Furthermore, MAPK activity decreases with age because of the increase
in MAPK phosphatase. Induction of
c-fos and
c-jun and AP-1 DNA binding activity
are concomitantly reduced (25). However, the MEK inhibitor, PD-098059,
does not inhibit DNA synthesis in aged hepatocytes, suggesting that the
age-related impairment in stimulated DNA synthesis may occur before E2F
activation and that signal transduction sequences other than the MAPK
pathway may be involved with stimulated DNA synthesis in the aged
hepatocytes.
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ACKNOWLEDGEMENTS |
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We thank Dr. Alan R. Saltiel of Parke-Davis Pharmaceutical Research Division for his generous gift of PD-098059 and Gloria Dunnigan for typing the manuscript.
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FOOTNOTES |
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Address for reprint requests: G. S. Roth, Molecular Physiology and Genetics Section, Laboratory of Cellular and Molecular Biology, Gerontology Research Center, 5600 Nathan Shock Dr., Baltimore, MD 21224.
Received 16 October 1997; accepted in final form 7 April 1998.
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