2 Veterans Affairs Medical Center, Departments of 1 Internal Medicine and 3 Biochemistry and Molecular Biology, and 4 Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan 48201
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ABSTRACT |
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Although in Fischer 344 rats aging is found to be associated with increased gastric mucosal proliferative activity, little is known about specific changes in the regulatory mechanisms of this process. To determine whether changes in cell cycling events could partly contribute to the age-related rise in gastric mucosal proliferative activity, the present investigation examines changes in cyclin-dependent kinase (Cdk2) activity and the regulation of this process in the gastric mucosa of Fischer 344 rats aged 4 (young), 13 (middle aged), and 24 (old) mo. We observed that aging is associated with a progressive rise in activity and protein levels of Cdk2 in the gastric mucosa. This is also found to be accompanied by a concomitant increase in cyclin E but not cyclin D1 levels. On the other hand, the levels of p21Waf1/Cip1 (total as well as the fraction associated with Cdk2), a nuclear protein that is known to inhibit different cyclin-Cdk complexes, are found to decline in the gastric mucosa with advancing age. In contrast, with aging, there was a steady rise in p53 levels in the gastric mucosa. We have also observed that the levels of phosphorylated retinoblastoma protein, a form that participates in regulating progression through the S phase, are markedly elevated in the gastric mucosa of aged rats. In conclusion, our data suggest that, in the gastric mucosa, aging enhances transition of G1 to S phase as well as progression through the S phase of the cell cycle. However, the age-related decline in p21Waf1/Cip1 in the gastric mucosa appears to be independent of p53 status.
cyclins; cyclin-dependent kinases; p21; p53; retinoblastoma
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INTRODUCTION |
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RESULTS FROM THIS AND OTHER laboratories have demonstrated in the Fischer 344 rat model that aging is associated with increased mucosal proliferative activity in various tissues of the gastrointestinal tract, including the stomach (1, 18, 19, 29, 31, 32, 34, 35). In the gastric mucosa, increased proliferative processes were evidenced by enhanced labeling index (34), DNA synthesis (10, 29), and thymidine kinase (29, 32) and ornithine decarboxylase (10) activities, each of which is known to be induced during the S phase of the cell cycle. Together, the results suggest that aging enhances progression of gastric mucosal cells through G1 into S phase of the cell cycle.
However, progression of mammalian cells through different cell cycle phases is a highly regulated process. This is largely controlled by cyclins, the regulatory units for cyclin-dependent kinases (Cdks) (22, 42). Cdks are a highly conserved family of serine/threonine protein kinases that phosphorylate several molecules involved in cell cycle progression. Binding of cyclins to Cdks activates their intrinsic kinase activity, triggering events that induce progression of cells through different phases of the cell cycle (34, 40, 46). Moreover, activation of Cdks depends on the binding of a specific cyclin (46). For example, whereas Cdk4 and Cdk6 require D-type cyclins for their activation (43), Cdk2, which is involved in G1/S transition, is activated by cyclin E (6, 17). In addition to cyclins, Cdks are also regulated by a class of Cdk inhibitors. Of these, p21Waf1/Cip1 (p21) and p27kip1 (p27), which have been studied extensively, are considered to be universal inhibitors of different cyclin-Cdk complexes (16, 44).
Although our earlier studies have demonstrated that aging is associated with a rise in gastric mucosal proliferative activity in Fischer 344 rats (34, 35), regulation of this process is poorly understood. In view of the fact that several parameters of S phase of the cell cycle, including labeling index and the rate of DNA synthesis, are higher in the gastric mucosa of aged than in young rats, we postulated that aging may stimulate G1/S phase transition in the gastric mucosa. To test this hypothesis, we examined the age-related changes in Cdk2 activity and regulation of this process. In addition, involvement of retinoblastoma (Rb) protein in regulating gastric mucosal cell cycle during advancing age was also investigated.
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METHODS |
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Reagents.
Antibodies (monoclonal or polyclonal) to Cdk2, cyclin D1, and
p21Waf1/Cip1 and p53 were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal
antibodies to cyclin E were from Upstate Biotechnology (Lake Placid,
NY). Polyclonal antibodies to phosophoylated Rb (Ser-795) were from New
England Biolabs (Beverly, MA). Goat anti-mouse or rabbit IgG conjugated
with horseradish peroxidase and the enhanced chemiluminescence (ECL)
system were obtained from Amersham (Arlington Heights, IL). Immobilon-P
nylon membrane was from Millipore (Bedford, MA), and X-Omat AR film was
from Eastman Kodak (Rochester, NY). [-32P]ATP (6,000 Ci/mmol) was obtained from NEN Life Science (Boston, MA). Histone H1
was from Sigma (St. Louis, MO). Concentrated protein assay dye reagent
was from Bio-Rad Laboratories (Hercules, CA). Molecular weight markers
were from GIBCO BRL (Grand Island, NY). Protein A-Sepharose beads were
from Pharmacia (Piscataway, NJ). All other reagents were of molecular
biology grade and were purchased from either Sigma or Fisher Scientific.
Animals and collection of tissues.
Male Fischer 344 rats aged 4 (young), 13 (middle-aged), and 24 (old) mo
were used. Four to five animals from each age group were utilized in
this investigation. The animals were purchased from the National
Institute on Aging (Bethesda, MD) 1 mo before the experiment. During
this period, they had access to Purina rat chow and water ad libitum.
In all experiments, the animals were fasted overnight before being
killed. The stomach was removed, cut along the greater curvature, and
rinsed thoroughly with cold normal saline, and mucosa from the oxyntic
gland area (hereafter referred to as gastric mucosa) was obtained by
scraping with glass slides. Mucosal scrapings were either processed
immediately or frozen in liquid nitrogen and stored in small aliquots
at 90°C.
Processing of mucosal scrapings. Aliquots of gastric mucosal scrapings were homogenized in a homogenizing buffer (10 mM HEPES, pH 7.2, 150 mM NaCl, 1 mM MgCl2, 2.5 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 1 mM of 1:10 phenanthroline, 2.5 mM EDTA, 25 µg/ml aprotinin, 25 µg/ml leupeptin, 50 µg/ml soybean trypsin inhibitor, 0.5% Triton X-100, and 0.5% NP-40). The homogenates were rotated for 30 min at 4°C and subsequently centrifuged at 11,000 g for 15 min at 4°C. The supernatants were used in all experiments. Protein concentration was measured by the Bio-Rad protein assay kit.
Western immunoblot analysis. This was performed essentially according to our standard protocol (33). In all analyses, protein concentration was standardized among the samples. Briefly, aliquots of mucosal samples containing either 50 or 100 µg protein were separated by SDS-PAGE and then electroblotted to Immobilon-P nylon membranes. The membranes were blocked overnight with 5% nonfat dried milk or 1% BSA in TBST buffer (20 mM Tris, pH 7.6, 100 mM NaCl, and 0.1% Tween 20), followed by 3 h of incubation with the primary antibody (1:1,000 to ~1:1,500 dilution) in TBST buffer containing 5% nonfat dried milk or 1% BSA at room temperature. After they were washed three times with TBST buffer, the membranes were incubated with a horseradish peroxidase-conjugated goat anti-mouse or rabbit IgG as a second antibody (1:5,000 dilution) for 1 h at room temperature. The reactions were visualized using the ECL detection system. For determination of p21Waf1/Cip1 bound to Cdk2, 1,500 µg of protein lysates were immunoprecipitated with 1 µg monoclonal anti-Cdk2 antibody, resolved by 15% SDS-PAGE, and transferred to the membranes. The membranes were then probed with polyclonal anti-p21 antibody. Signals on the blots were visualized by autoradiography and quantitated by densitometry using ImageQuant image analysis system (Storm Optical Scanner, Molecular Dynamics, Sunnyvale, CA). All of the Western immunoblots were performed at least three times using different rats for each age group. Similar results were obtained in all experiments.
Cdk2 activity.
The enzyme activity was determined essentially according to the
procedure described by Evers et al. (9). Briefly, aliquots of mucosal
lysate containing 500 µg of protein were subjected to
immunoprecipitation with 1 µg anti-Cdk2 antibody at 4°C for 3 h.
Immune complexes were recovered with protein A-Sepharose beads, washed
three times with 50 mM Tris (pH 7.6), 150 mM NaCl, and 0.5% Tween 20, and then washed twice with kinase washing buffer [25 mM HEPES, pH
7.4, 10 mM MgCl2, 1 mM
dithiothreitol (DTT), and 0.5 mM
Na3VO4].
The kinase reaction was performed with 25 µl of kinase buffer (25 mM
HEPES, pH 7.4, 10 mM MgCl2, 1 mM
DTT, 0.5 mM
Na3VO4,
5 µg histone H1, and 40 µM ATP) containing 10 µCi [-32P]ATP at
30°C for 30 min. The reaction was stopped by the addition of
2× sample loading buffer (125 mM Tris, pH 6.8, 4% SDS, 10% glycerol, 4%
-mercaptoethanol, and 0.02% bromphenol blue). The samples were boiled for 4 min and resolved by 12% SDS-PAGE. The gels
were dried, and then signals were visualized by autoradiography and
quantitated by densitometry. Cdk2 kinase assay was performed three to
four times using mucosal samples from different rats for each age group.
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RESULTS |
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The first set of experiments were performed to determine whether
activation of Cdk2, an enzyme required for progression of cells through
late G1 into S phase, was affected
by aging. We found Cdk2 activity in the gastric mucosa to rise
progressively with advancing age (Fig. 1).
The enzyme activity in the gastric mucosa of 13- and 24-mo-old rats was
increased by 120% and 420%, respectively, over their 4-mo-old
counterparts (Fig. 1).
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To determine whether the age-related rise in Cdk2 activity in the
gastric mucosa could partly be due to increased levels of the enzyme,
the relative concentration of Cdk2 was analyzed by Western immunoblot.
As has been observed for the enzyme activity, protein levels of Cdk2 in
the gastric mucosa also rose steadily with advancing age, revealing a
75% and 125% increase, respectively, in 13- and 24-mo-old rats over
their 4-mo-old counterparts (Fig. 2).
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Because cyclins are essential for activation of Cdks, levels of cyclin
E and D1 in the gastric mucosa of 4-, 13-, and 24-mo-old rats were next
examined by Western immunoblot analysis. As expected, levels of cyclin
E, which is necessary for activation of Cdk2 (5, 13), were found to be
75% and 220% higher, respectively, in 13- and 24-mo-old rats compared
with 4-mo-old animals (Fig. 3). In
contrast, no apparent change in cyclin D1 levels in the gastric mucosa
was observed among different age groups (Fig.
4).
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p21Waf1/Cip1 is considered to be
one of the universal inhibitors of different cyclin-Cdk complexes (16,
44). Therefore, the next experiment was performed to determine whether
the age-related rise in gastric mucosal Cdk2 activity could be partly
attributable to reduction in
p21Waf1/Cip1 levels in the gastric
mucosa. Totals as well as the fractions of
p21Waf1/Cip1 bound to mucosal Cdk2
were examined by Western immunoblot in all three groups of rats.
Indeed, we observed that levels of both forms of
p21Waf1/Cip1 in the gastric mucosa
of 24-mo-old rats were 30-50% lower than in their younger
counterparts (Figs. 5 and
6).
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It has been demonstrated that
p21Waf1/Cip1 expression can be
either dependent on or independent of the p53 status, the tumor
suppressor protein that is shown to be required for transactivation of
a number of growth regulatory genes, including
p21Waf1/Cip1 (7, 11). Therefore,
to determine the relationship between p53 and
p21Waf1/Cip1 in the gastric mucosa
during aging, levels of p53 protein were analyzed by Western immunoblot
in the gastric mucosa from all three age groups. Our results showed an
inverse relationship between these two proteins in that the age-related
decrease in p21Waf1/Cip1
expression (Fig. 5) was associated with a concomitant rise in p53
levels in the gastric mucosa (Fig. 7). The
relative concentration of p53 protein in the gastric mucosa of 13- and
24-mo-old rats was found to be 60% and 150%, higher, respectively,
than in 4-mo-old animals (Fig. 7).
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The product of Rb gene, which encodes a nuclear phosphoprotein, is
known to be differentially phosphorylated during the cell cycle (20,
42). Because phosphorylation of Rb, catalyzed by Cdks, results in
progression of the cell cycle through the S phase, levels of
phosphorylated Rb protein were measured in the gastric mucosa of all
three groups of rats. Results revealed that maximal stimulation in Cdk2
activity, which was observed in 24-mo-old rats, was also accompanied by
an analogous rise in phosphorylated Rb protein levels in the gastric
mucosa (Fig. 8). The relative concentration
of phosphorylated Rb protein in the gastric mucosa of 24-mo-old rats
was 125% higher than in their 4-mo-old counterparts (Fig. 8). However,
in 13-mo-old rats, phosphorylated Rb protein levels were only
marginally increased (~20%), compared with 4-mo-old animals (Fig.
8).
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Finally, it should be stated that, although a representative autoradiograph from one experiment was presented, each analysis was repeated three to four times with mucosal samples from four to five rats in each age group.
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DISCUSSION |
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Cells of the gastrointestinal mucosa undergo a constant process of renewal, which in normal adults reflects a balance between proliferation of precursor cells and exfoliation of surface cells (4, 23, 24). A detailed knowledge of mucosal cell proliferation and kinetics at different stages of life is, therefore, of paramount importance for a better understanding of the normal aging process as well as of many gastrointestinal diseases that represent disorders of tissue growth (5, 8, 24, 26, 29, 48). In general, many gastrointestinal dysfunctions, including malignancies, increase with advancing age, which in itself is associated with marked alterations in the structural and functional properties of the gastrointestinal tract (5, 12).
Previous studies have demonstrated that the rate of gastrointestinal mucosal cell proliferation changes throughout life (21, 27, 28, 30). Earlier, we reported that gastric mucosal proliferative activity in rats remains elevated during the first 2 wk of postnatal life and then decreases dramatically over the next 2-3 wk (36). Conversely, morphological as well as biochemical studies from this and other laboratories indicate that aging is associated with increased gastric as well as small and large intestinal mucosal proliferative activity (1, 18, 19, 29, 31, 32, 34, 35). At least in the gastric mucosa, the age-related rise in proliferative activity could not be attributed to increased responsiveness of gastrin, bombesin, or epidermal growth factor (EGF), each of which is known to stimulate gastrointestinal mucosal growth in young animals (21). In fact, we have demonstrated that aging is associated with a loss of responsiveness of the gastric mucosa to the trophic action of both gastrin and bombesin (32, 37), whereas EGF actually inhibits mucosal proliferative activity in aged rats (29).
Little is known about the responsible intracellular mechanisms for the age-related rise in mucosal proliferative activity. Our current data demonstrate, for the first time, that, at least in the gastric mucosa, this could be partly attributed to increased G1 to S phase transition. This inference is based on the observation that Cdk2 activity in the gastric mucosa increases steadily with advancing age. Because progression through the late G1 into S phase requires activation of Cdk2 (6, 17), our observation of a substantially higher Cdk2 activity in the gastric mucosa of 13- and 24-mo-old rats than in their 4-mo -old counterparts suggests that aging accelerates the late G1 phase of the cell cycle.
Although multiple mechanisms are involved in regulating Cdk2 activity, formation of Cdk-cyclin complex is considered to be one of the primary causes for activation of the enzyme (22, 42). In view of the fact that activation of Cdk2 requires cyclin E, the parallelism between Cdk2 activity and cyclin E levels in the gastric mucosa during aging suggests a critical role for cyclin E in regulating the age-related rise in Cdk2 activity in the gastric mucosa. Each cyclin is thought to regulate a particular cell cycle phase by binding to and activating a specific Cdk (38, 40, 43, 46). Therefore, a steady rise in Cdk2 activity and its regulatory protein cyclin E levels in the gastric mucosa during advancing age also supports our contention that aging is associated with enhanced progression of gastric mucosal cells through the late G1 phase. This inference is further supported by the observation that, in the gastric mucosa, expression of D1 cyclin, which is involved in regulating Cdk4 and Cdk6 but not Cdk2 (43), is unaffected by aging. In addition to these observations, we have noted that the age-related rise in Cdk activity is also accompanied by a concomitant increase in protein levels of Cdk2, suggesting that increased levels of the enzyme could also contribute to the steady rise in its activity in the gastric mucosa during advancing age.
Among other factors that may modulate Cdk2 activity, p21Waf1/Cip1, which is one of the universal inhibitors of the cyclin-Cdk complexes (16, 44), can be considered a prime candidate. p21Waf1/Cip1, a 21-kDa protein, has been shown to inhibit not only Cdks but also proliferating cell nuclear antigen (13, 14, 39) by forming a tetrameric complex. Formation of this complex inhibits cells from entering and progressing through the S phase of the cell cycle (15, 47). Thus overexpression of p21Waf1/Cip1 is likely to result in inhibition of cell proliferation by attenuating various Cdks involved in cell cycling, whereas an opposite phenomenon can be expected with diminished expression of this protein. Our current observation of a inverse relationship between Cdk2 activity and p21Waf1/Cip1 levels (total as well as the fraction bound to Cdk2) in the gastric mucosa during aging suggests that decreased inhibition of Cdk2 activity resulting from lowered levels of p21Waf1/Cip1 in the gastric mucosa during aging has also contributed to the age-related rise in mucosal Cdk2 activity.
However, the decreased expression of p21Waf1/Cip1 in the gastric mucosa of aged rats could not be attributed to p53, a transcription factor that has been shown to participate in transactivation of a number of growth regulatory genes, including p21Waf1/Cip1 (7, 11). Our observation that protein levels of p53 and p21Waf1/Cip1 in the gastric mucosa are inversely related during advancing age suggests that, at least in this tissue, the age-related changes in p21Waf1/Cip1 appear to be regulated through a p53-independent mechanism. A similar phenomenon has also been noted in other conditions (11). For example, in senescent human diploid fibroblasts, the increase in p21Waf1/Cip1 has been shown to be independent of p53 status (2, 41, 45).
p21Waf1/Cip1 has also been shown to inhibit phosphorylation of the Rb gene product (25), a nuclear protein that is progressively phosphorylated and dephosphorylated through the cell cycle (20, 42). In its hypophosphorylated state during G1 phase, Rb binds a number of cellular proteins, most notably the E2F transcription factor, resulting in inhibition of cell cycling. On the other hand, phosphorylation of Rb, catalyzed by Cdks, causes dissociation of the transcription factor bound to Rb, resulting in enhancement of progression of the cell cycle through the S phase. The current observation that the level of phosphorylated Rb is greater in the gastric mucosa of aged than in young rats further suggests that aging also enhances progression through the S phase of the cell cycle in the gastric mucosa.
In conclusion, our current data demonstrate that aging is associated with induction of a G1 checkpoint in the gastric mucosa, as evidenced by a rise in activity and expression of Cdk2 as well as its regulatory protein, cyclin E. These changes are accompanied by decreased expression of p21Waf1/Cip1, a universal inhibitor of Cdks. Moreover, the fact that the levels of phosphorylated Rb in the gastric mucosa are greatly elevated in aged rats also suggests that aging may stimulate progression of mucosal cells through the S phase of the cell cycle. The age-related decline in p21Waf1/Cip1 in the gastric mucosa appears to be independent of the p53 status.
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ACKNOWLEDGEMENTS |
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This work was supported by grants from the National Institute on Aging (AG-14343) and the Department of Veterans Affairs.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. P. N. Majumdar, Research Service 151, VA Medical Center, 4646 John R, Detroit, MI 48201 (E-mail: a.majumdar{at}wayne.edu).
Received 17 June 1999; accepted in final form 31 July 1999.
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