Section of Physiology, Cornell University, Ithaca, New York 14853
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ABSTRACT |
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Using the conditionally immortalized human cell line tsFHI, we have investigated the role of cyclin-dependent kinase inhibitors (CKIs) in intestinal epithelial cell differentiation. Expression of cyclins, cyclin-dependent kinases (Cdk), and CKIs was examined under conditions promoting growth, growth arrest, or expression of differentiated traits. Formation of complexes among cell cycle regulatory proteins and their kinase activities were also investigated. The tsFHI cells express three CKIs: p16, p21, and p27. With differentiation, p21 and p27 were strongly induced, but with different kinetics: the p21 increase was rapid but transient and the p27 increase was delayed but sustained. Our results suggest that the function of p16 is primarily to inhibit cyclin D-associated kinases, making tsFHI cells dependent on cyclin E-Cdk2 for pRb phosphorylation and G1/S progression. Furthermore, they indicate that p21 is the main CKI involved in irreversible growth arrest during the early stages of cell differentiation in association with D-type cyclins, cyclin E, and Cdk2, whereas p27 may induce or stabilize expression of differentiated traits acting independently of cyclin-Cdk function.
tsFHI; cyclin-dependent kinase inhibitors
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INTRODUCTION |
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NORMAL DEVELOPMENT and function of the small intestinal epithelium depend on a tight regulation of proliferation and differentiation in the crypts. Although the morphological and self-renewing properties of the crypt cells have been well characterized in animal models (15, 16), the mechanisms controlling their proliferation and commitment to differentiation into different types of functional villus cells are little understood. The development of tissue culture models centered on conditionally immortalized animal (12, 34, 47) or human (37) crypt cells has opened new avenues of investigation into the molecular aspects of intestinal cell differentiation. We recently established a clonal cell line of SV40 T-antigen (SV40 T-Ag)-transformed human intestinal epithelial cells (tsFHI cells), in which cell differentiation can be induced by a simple temperature shift and further modulated by a variety of culture medium supplements (37). Two phases in the differentiation process could be experimentally defined: 1) an early phase (up to 2-3 days after the temperature shift), characterized by loss of the SV40 T-Ag, marked reduction in p53 expression, and irreversible loss of proliferative potential, but without significant changes in differentiation markers, and 2) a later phase, during which brush-border enzymes such as dipeptidyl peptidase IV (DPPIV), aminopeptidase N (APN), and sucrase-isomaltase are rapidly induced together with marked morphological alterations (increase in cell size, appearance of a dense layer of apical microvilli, and formation of tight junctions). Another important characteristic of this model is that growth inhibition and cell differentiation can, to some extent, be investigated as separate phenomena, allowing us to identify and specifically focus on regulatory mechanisms involved in cell differentiation alone.
In preliminary experiments we have also observed that expression of p21(WAF1/Cip1) (p21) was markedly increased in tsFHI cells within hours of a shift to the nonpermissive temperature (39°C), preceding the appearance of morphological and functional markers of differentiation by at least 1-2 days (37). This was reminiscent of similar observations made using different cell types in vivo and in vitro, pointing to an important role of this (and other) cyclin-dependent kinase inhibitors (CKIs) not only in growth arrest but also in modulation of cell differentiation. CKIs are relatively small intracellular proteins that bind to cyclins or cyclin-cyclin-dependent kinase (Cdk) complexes and inhibit their activity (41, 42). The prototype, p21, has been discovered because of its stimulation by wild-type but not mutant p53 (10), its increased expression and growth-inhibitory activity in senescent human cells (32), and its ability to associate with and inactivate most cyclin-Cdk complexes (20, 21, 48). p21 has also been proposed as a key mediator of p53 functions after chromosomal DNA damage (27, 46). The ability of p21 to block cell cycle progression has been related to inhibition of retinoblastoma protein (pRb) phosphorylation (31), but p21 is also known to complex with the proliferating cell nuclear antigen (PCNA), a protein necessary for DNA elongation after the priming step (45, 49). The Cdk and PCNA inhibitory activities of p21 are functionally independent and reside in the amino- and carboxy-terminal domains, respectively (5, 26). Recent evidence has suggested that p21 also plays an important role in terminal cell differentiation through p53-independent pathways (18, 22, 43). The relevance of these in vitro findings is supported by studies conducted in p53-deficient mice, where the developmental pattern of p21 expression has been correlated with differentiation of multiple cell lineages, including skeletal muscle, cartilage, skin, and nasal epithelium (33).
Other proteins with functions similar to p21 have also been identified and classified in two families according to their homology: the Cip/Kip family includes p21, p27, and p57 (36); the Ink4 family includes p16, p15, p18, and p19 (42). A distinguishing feature of the Cip/Kip family is that its members have broad target specificity. They can effectively target Cdk2 in complexes with cyclin A and E, as well as Cdk4 and Cdk6 in complexes with cyclins D1, D2, and D3 (42). In contrast, the Ink4 family members have more restricted targets: Cdk4 and Cdk6 (13, 40).
Previous studies aimed at investigating p21 expression in the intestinal tract in vivo have produced sometimes conflicting or inconclusive results, and the role of this and other CKIs in intestinal cell growth regulation or differentiation has yet to be firmly established. In the normal human colorectal epithelium, p21 was undetectable in the lower one-third of the crypts, where the proliferation marker Ki67 was expressed, leading to the conclusion that p21 expression is correlated with cessation of proliferation, rather than with differentiation (11). In contrast, in our previous study we could detect p21 in only a fraction of upper-crypt and lower-villus cells in fetal and adult human intestine (37). This discrepancy may reflect differences in the specific antibodies used for immunolocalization or in the expression patterns of the p21 protein along the length of the intestinal tract. With use of in situ hybridization, and in the mouse, the highest levels of p21 mRNA were detected in epithelial cells lining the uppermost region of the crypts and the lower one-third of the villi in the duodenum and also in a few cells at the base of the crypts tentatively identified as Paneth cells (14). In the mouse colon and in all other tissues examined (tongue, cervix, and hair follicles), strong hybridization was observed only in cells that had ceased proliferation and begun the process of terminal differentiation (14).
To further investigate the potential role of p21 and other CKIs in intestinal cell dynamics, in the present study we have examined their expression, together with that of other related cell cycle regulatory proteins such as cyclins and Cdks, in tsFHI cells cultured under conditions promoting cell growth, growth arrest without significant increase in differentiation markers, or maximal cell differentiation. Our results have implicated p21 and p27 in the intestinal differentiation process, but with temporally and functionally distinct roles.
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MATERIALS AND METHODS |
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Cell culture.
tsFHI, a conditionally immortalized intestinal epithelial cell line
derived from fetal human intestine at 19 wk of gestation, was
established and characterized by Quaroni and Beaulieu (37). Two media
were used for their culture: a growth medium (GM) and a differentiation
medium (DMD). For maximal cell growth, tsFHI cells were routinely
cultured in a humidified incubator with 6% CO2 at 31°C in GM, consisting
of Opti-MEM I (GIBCO-BRL, Grand Island, NY) supplemented with 4% fetal
bovine serum (FBS), 10 ng/ml epidermal growth factor, 2 mM glutamine, 2 mM L-alanyl-glutamine (GlutaMAX
I), 10 mM HEPES, 50 U/ml penicillin, and 50 µg/ml streptomycin. They
were passaged every 12-15 days when about two-thirds confluent. Their differentiation was induced by culture in DMD, consisting of DMEM
with 4.5 g/l glucose (DMEM) supplemented with 7% FBS, 50 ng/ml
dexamethasone, 2 mM glutamine, 2 mM GlutaMAX I, 10 mM HEPES, 50 U/ml
penicillin, and 50 µg/ml streptomycin. Six different experimental
culture conditions, promoting growth arrest and/or differentiation
(37), were used in this study by altering the culture medium (GM or
DMD) or the incubator temperature (31 or 39°C) and by inhibiting
cell growth with transforming growth
factor-1 (TGF-
1):
1) 31°C in GM (maximal cell
growth), 2) 31°C in GM-1 ng/ml
TGF-
1 for 7-14 days (total
but entirely reversible growth arrest),
3) 31°C in DMD for 7 days (total
growth arrest, reversible in only a small fraction of cells),
4) 39°C in DMD for 1 day
(expression of early growth arrest-related genes),
5) 39°C in DMD for 3 days (expression of growth arrest-related genes), and
6) 39°C in DMD for 7 days
(irreversible growth arrest and marked increase in cell differentiation markers).
Sources of antibodies. The following affinity-purified rabbit polyclonal and mouse monoclonal antibodies (and corresponding blocking peptides) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and used for immunoprecipitation or Western blotting: rabbit anti-p15 [C-20 (sc-612)] against a peptide corresponding to amino acids 118-137 mapping at the carboxy terminus of the human p15; rabbit anti-p16 [C-20 (sc-468)] against a peptide corresponding to amino acids 137-156 mapping at the carboxy terminus of the human p16; rabbit anti-p18 [N-20 (sc-865)] against a peptide corresponding to amino acids 2-21 mapping at the amino terminus of the human p18; rabbit anti-p19 [M-167 (sc-1063)] against a glutathione S-transferase-tagged fusion protein containing sequences corresponding to amino acids 1-167 representing the full length of mouse p19; rabbit anti-p21 [C-19 (sc-397)] against a peptide corresponding to amino acids 146-164 mapping at the carboxy terminus of the human p21; rabbit anti-p27 [C-19 (sc-528)] against a peptide corresponding to amino acids 181-198 at the carboxy terminus of the human p27; rabbit anti-Kip2 p57 [C-20 (sc-1040)] against a peptide corresponding to amino acids 286-305 mapping at the carboxy terminus of the human Kip2 p57; rabbit anti-Cdk2 [M2 (sc-163)] against a peptide corresponding to amino acids 283-298 mapping at the carboxy terminus of the human Cdk2; rabbit anti-Cdk4 [H-22 (sc-601)] against a peptide corresponding to amino acids 282-303 mapping at the carboxy terminus of the human Cdk4; rabbit anti-Cdk6 [C-21 (sc-177)] against a peptide corresponding to amino acids 306-326 mapping at the carboxy terminus of the human Cdk6; anti-cyclin D1 [R-124 (sc-6281)], a mouse monoclonal antibody against the full-length (1-295) human cyclin D1; rabbit anti-cyclin D2 [C-17 (sc-181)] against a peptide corresponding to amino acids 274-289 mapping at the carboxy terminus of human cyclin D2; rabbit anti-cyclin D3 [C-16 (sc-182)] against a peptide corresponding to amino acids 277-292 mapping at the carboxy terminus of the human cyclin D3; anti-PCNA [PC10 (sc-56)], a mouse monoclonal antibody against recombinant PCNA; anti-cyclin A [BF683 (sc-239)], a mouse monoclonal antibody against the recombinant human cyclin A protein; anti-cyclin E [HE111 (sc-248) and HE12 (sc-247)], mouse monoclonal antibodies produced against recombinant human cyclin E protein; rabbit anti-pRB [C-15 (sc-50)] against a peptide corresponding to amino acids 914-928 mapping at the carboxy terminus of the human pRb p110; and anti-p53 [DO-1 (sc-126)], a mouse monoclonal antibody against human wild-type p53. The mouse monoclonal antibody against SV40 T-Ag [Pab101 (clone 412)] was obtained from the American Type Culture Collection. Donkey anti-rabbit or sheep anti-mouse antibodies conjugated with horseradish peroxidase were purchased from Amersham Life Science (Arlington Heights, IL). Protein G Plus/Protein A-agarose mixtures were purchased from Oncogene Research Products (Cambridge, MA).
Indirect immunofluorescence staining. Cells, grown in 35-mm dishes, were fixed with 3% paraformaldehyde and stained by the indirect immunofluorescence method, as previously described (37). The secondary antibodies were FITC- or rhodamine-conjugated goat anti-mouse or donkey anti-rabbit IgG (Boehringer Mannheim, Indianapolis, IN) and diluted 1:25 in PBS. DNA was stained with 4',6-diamidino-2-phenylindole at 2 mg/ml for 4 min. Negative controls included nonimmune mouse serum (diluted 1:50) in place of the primary antibody or PBS in place of the secondary antibody. The cells were mounted with glycerol-PBS (9:1) plus 2.5% 1,4-diazabicyclo-[2.2.2]octane. Stained cells were examined with a Zeiss Axiovert 10 microscope equipped with epifluorescence optics.
Immunoprecipitation. For preparation of total cell lysates, cells grown in 100-mm-diameter dishes under the desired conditions were washed twice with ice-cold PBS, scraped with a rubber policeman into PBS containing 1 mM phenylmethylsulfonyl fluoride, and centrifuged at 2,500 rpm at 4°C for 15 min. The supernatants were removed, and the pellets were lysed in a lysis buffer containing 50 mM Tris · Cl, pH 7.5, 150 mM NaCl, 50 mM NaF, 1% NP-40, 1 mM dithiothreitol, 0.1 mM sodium vanadate, and the protease inhibitors leupeptin (25 µg/ml), aprotinin (25 µg/ml), soybean trypsin inhibitor (10 µg/ml), benzamidine (1 mM), and phenylmethylsulfonyl fluoride (1 mM). The cell suspensions were sonicated for 5 s twice at setting 7 with a cell disrupter (model 200, Branson Sonifier, equipped with a microtip), incubated on ice for 30 min (mixing every 5 min), and cleared by centrifugation (10,000 g, 5 min, 4°C). DNA concentrations were measured in the cell lysates by use of a fluorometer and the Hoechst method (Pharmacia) and used to calculate the cell concentration (cells/ml lysate). The DNA content per cell was determined using separate samples in which the cell concentration was determined by hemocytometer counting. In each Western blot or immunoprecipitation experiment, samples of cells subjected to different experimental conditions were standardized by using volumes of cell lysates containing the same amount of DNA. Primary antibody at a concentration of 1 µg/ml was added to each cell lysate and incubated at 4°C for 2 h (for kinase assays) or for 16 h (for Western blot analysis). At the end of each incubation, 30 µl of Protein G Plus/Protein A-agarose mixture were added and incubated at 4°C for 1 h. For immunoblotting, the immunocomplexes were washed four times with the lysis buffer and resuspended in SDS-PAGE sample buffer. For kinase assays, the immunocomplexes were washed twice with the lysis buffer and once with the kinase buffer and assayed for kinase activity.
Immunoblotting. Immunoprecipitates or total cell lysates solubilized in SDS-PAGE sample buffer were subjected to SDS-PAGE, and Western blotting was performed essentially as described previously (3). Three different gels were used to separate proteins: 7.5% for pRb; 15% for p15, p16, p18, and p19; and 12% for the other proteins. After electrophoresis, proteins were electrically transferred onto nitrocellulose membranes (High-Bond nitrocellulose, Amersham) with use of a transblot system (Bio-Rad, Hercules, CA) at 100 V, 5°C, for 90 min. The membranes were blocked at 4°C overnight in blocking buffer containing PBS, 0.1% Tween 20, and 3% BSA, incubated with primary antibody diluted in blocking buffer at room temperature for 2 h, and washed three times in washing buffer containing PBS and 0.1% Tween 20. Appropriate secondary antibodies (1:3,000 dilution of horseradish peroxidase-linked sheep anti-mouse immunoglobulin or donkey anti-rabbit immunoglobulin, Amersham) were incubated with membranes for 1 h at room temperature. Specific proteins were detected using an enhanced chemiluminescence system (ECL protocol, Amersham Life Science). When it was necessary, the intensities of protein bands were analyzed using a densitometer (Ultroscan, LKB, Bromma, Sweden). As required, the membranes were stripped, blocked, and reprobed with different antibodies (ECL protocol, Amersham Life Science).
Enzyme assays. DPPIV and APN activities were determined as previously described (37).
For determination of Cdk2-associated kinase activity, immunoprecipitates were washed twice with ice-cold lysis buffer and then once with ice-cold kinase buffer containing 50 mM HEPES, pH 7.0, 10 mM MgCl2, and 1 mM dithiothreitol. Kinase reactions were started by incubating the washed immunoprecipitates at 30°C in the kinase buffer containing 20 µM ATP, 5 µCi [RNase protection assay. Total cellular RNA was isolated from tsFHI cells using an RNeasy kit (Qiagen, Chatsworth, CA). The integrity of the RNA was verified by ethidium bromide staining, and the quantity was determined spectrophotometrically. The mRNA levels of several genes were simultaneously analyzed using the Multi-RiboQuant RNase protection assay (RPA) kit (Pharminigen, San Diego, CA). Three sets of the riboprobe templates (hcc-1, hcc-2, and hcyc-1) were purchased from Pharminigen and used to transcribe 21 antisense RNA probes, allowing us to investigate the mRNA levels of cyclin A, cyclin B, cyclin C, cyclin D1, cyclin D3, Cdk3, p57, p27, p21, p19, p18, p16, p14/p15, and p53. Two housekeeping genes, L32 and glyceraldehyde-3-phosphate dehydrogenase, were also included in all three sets of templates as internal controls. From each transcription reaction, 4 × 105 cpm were used to hybridize 5 µg of total RNA. The RPA procedures were as described in the manufacturer's protocol (Pharminigen). The protected fragments were finally separated on 6% precast sequencing gels (Stratagene, La Jolla, CA). Then gels were fixed, dried, and exposed to X-ray film.
Statistical analysis. ANOVA was used as a statistical test. Values are means ± SE and were considered significant when P < 0.05.
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RESULTS |
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Expression of p21 in tsFHI cells.
We previously reported that tsFHI cells, under optimal growth
conditions at 31°C, express very low levels of p21, and this CKI is
rapidly increased in cells induced to differentiate by a shift to
39°C in DMD (37). Immunofluorescence staining revealed two
important characteristics of p21 expression in these cells (Fig.
1). When cells were
cultured at 31°C in GM, staining was restricted to a small fraction
of intensely stained nuclei (Fig. 1, A
and B), and neither the frequency
nor the intensity of the positive nuclei was increased by growth arrest
in the presence of TGF-1 (Fig.
1, C and
D). A similar observation was made
with cells in which growth was arrested by culture in DMD at 31°C
(not shown). A shift to 39°C resulted in intense staining of
>90% of the nuclei within 1 day (Fig. 1,
E and
F); at later times the intensity of
staining declined markedly, but the frequency of positive nuclei did
not change significantly (Fig. 1, G
and H).
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Increased levels of p21, p27, and cyclins
D2 and D3 in
tsFHI cells induced to differentiate by a shift to 39°C.
Cell cycle progression is controlled by a complex interplay among
several classes of regulatory proteins, including cyclins, Cdks, and
CKIs. Thus our aim was to evaluate their relative protein levels in
tsFHI cells 1) under optimal growth
conditions (at 31°C in GM), 2)
growth arrested at the permissive temperature (by culture in DMD or in
the presence of TGF-1), or
3) further induced to differentiate
by culture at the nonpermissive temperature in DMD. We were also
interested in evaluating changes taking place early in differentiation
(1-3 days after the temperature switch) and in comparing these
changes with those associated with maximal expression of morphological
and functional markers of differentiation (
7 days after the
temperature switch).
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mRNA levels in tsFHI cells cultured at 39°C.
To gain insight into the mechanisms responsible for the changes in
protein level described above, we analyzed the mRNA levels of p16, p21,
p27, and other cell cycle regulatory proteins under the same six
culture conditions promoting growth, growth arrest, or differentiation.
The RPA we used allowed us to evaluate a large number of mRNAs in a
single experiment and proved much more sensitive than Northern blot
analysis. The results obtained, quantified by densitometric scanning,
are illustrated in Fig. 4, and their salient features are as follows: 1)
The p21 mRNA was induced three- to fourfold 1 day after a shift to
39°C, a change comparable to that observed for its corresponding
protein levels. 2) p27 and p16 mRNA
levels were relatively constant or declined slightly (in the case of
p16) after the temperature shift. 3)
The mRNA levels of cyclin A, cyclin B, cyclin C, and Cdk3 decreased
markedly in tsFHI cells cultured at 39°C (Fig. 3,
A and
B).
4) The cyclin D1 mRNA level was lower in cells
in which growth was arrested in DMD at 31°C and after 1 day at
39°C but increased with prolonged incubation at the higher
temperature. The levels of cyclin
D3 mRNA did not vary significantly
(Fig. 4B).
5) As observed for the corresponding
proteins, mRNAs for p57, p19, or p14/p15 were undetectable (data not
shown).
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Complex formation among CKIs, cyclins, and Cdks.
Because all known CKIs act in a stoichiometric rather than a catalytic
fashion, it was important to investigate the complexes p16, p21, and
p27 formed with other cell cycle regulatory proteins to determine their
potential functional role(s) in growth arrest or differentiation of
tsFHI cells. Immunoprecipitates obtained with antibodies directed
against the three CKIs, cyclin D, or cyclin E were sequentially
immunoblotted with antibodies specific for Cdk2, Cdk4, Cdk6, and
cyclins A, E, D1,
D2, and
D3. For simplicity, only selected
data are illustrated in Fig. 5. The main
results of these studies were as follows.
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pRb phosphorylation and kinase activities. One of the main functions of G1 cyclin-Cdk complexes is phosphorylation of the key regulatory protein pRb, an event releasing E2F and other transcriptional factors essential for cell cycle progression into the S phase (44). We have therefore investigated whether the changes in CKIs and cyclin-Cdk complexes noted above were accompanied by changes in the pRb expression level and/or its phosphorylation status. The two forms of pRb can be distinguished on SDS-PAGE gels, where hyperphosphorylated (inactive) pRb appears as a rather broad band of slower mobility and hypophosphorylated (active) pRb appears as a narrow band with faster mobility.
Both forms of pRb were observed in tsFHI cells growing in GM at 31°C (Fig. 7A, lane 1). Induction of cell differentiation at the nonpermissive temperature led to a rapid decline in the upper pRb band (Fig. 7A, lanes 2-8), which essentially disappeared by 36 h after the temperature switch. The cellular level of pRb itself (sum of the 2 bands) did not change significantly with growth arrest (not shown) or with cell differentiation. Figure 7A also shows that the SV40 T-Ag was rapidly degraded and essentially disappeared by 12-24 h at 39°C. The cellular level of p53 also decreased markedly at the nonpermissive temperature, but in a more gradual fashion (Fig. 7A). pRb remained in an underphosphorylated form with prolonged (up to 15 days) cell incubation at 39°C, during which time p53 levels declined further (Fig. 7B).
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DISCUSSION |
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Our main aim was to obtain evidence for a role of CKIs in induction or
modulation of intestinal epithelial cell differentiation. It was
therefore important to discriminate cellular alterations affecting
solely cell growth, and the general design of this study was based on
our previous observation that culture of tsFHI cells at the permissive
temperature in DMD or in the presence of
TGF-1 results in essentially
complete growth arrest without significant increases in functional or
morphological markers of differentiation (37). The presence of a
functional SV40 T-Ag in cells cultured at the permissive temperature
dictates caution in the interpretation of some of the results we have
obtained, since this protein is well known to interfere with pRb- and
p53-related functions. It is also known that p53 is an inducer of p21
expression (27, 46), and pRb may act as a negative regulator of p16
(28). Thus SV40 T-Ag-transformed cells may show rearrangements in
cyclin-Cdk-CKI complexes (48). Previous findings would predict
induction of p21 with restoration of p53 activity and a decline in p16
function when pRb is released from the inhibitory activity of the T-Ag. Although we cannot exclude the possibility that some of our
observations were the result of changes in SV40 T-Ag activity or
expression levels, the following considerations indicate that the main
conclusions of our study were not affected. tsFHI cells cultured at the
permissive temperature are without exception positive for T-Ag and p53
(37) (Fig. 1), yet p21 was restricted to a very small fraction of
intensely stained nuclei (Fig. 1, A
and B) and did not correlate with
p53 expression at the cellular level (Fig. 1,
I and
J). In cells shifted to 39°C the
temperature-sensitive T-Ag was almost entirely degraded after 8 h (Fig.
7A), potentially correlating with
induction of p21 starting at about the same time, but p53 levels were
still relatively high 2-3 days later when p21 started to decline
significantly, eventually to return to uninduced levels. Levels of p16
were relatively constant at both temperatures and, contrary to
expectations, increased slightly in the absence of a functional SV40
T-Ag (Fig. 2); the ability of this CKI to form complexes with Cdk4 was
also not significantly affected (Fig.
5C). Expression levels and activity
of p27 should not have been influenced by the SV40 T-Ag status, and in
fact the delayed induction of this CKI on temperature shift did not temporally correlate with T-Ag degradation or restoration of p53 and
pRb functions. A question that does remain open for future investigation is the mechanism(s) regulating p21 expression in tsFHI
cells. However, in the intestine in vivo, as in other tissues and cells
induced to differentiate, p21 was found to be induced by
p53-independent mechanisms (11, 14, 22, 33, 37), and the presence of
scattered intensely stained nuclei in tsFHI cells cultured at 31°C
would favor such an interpretation.
The results we have obtained seem to exclude a significant role for p21
or p27 in inhibition of tsFHI cell growth by
TGF-1. This is surprising,
because previous reports have implicated p15 (19) and p27 (39) as key
mediators of TGF-
in a variety of cell lines. Because tsFHI cells do
not express any detectable p15, this CKI could not be implicated in
their cell cycle regulation; in the case of p27, neither its expression
levels (Fig. 2) nor its ability to form complexes with cyclins and Cdks
(Fig. 5) was affected by TGF-
. p16 and p21 were also unaltered in
their cellular levels or association with Cdks and cyclins in
TGF-
-treated cells. It should be noted that although the growth
curves illustrated in our previous publication (Fig. 2 in Ref. 37)
demonstrated growth inhibition in the presence of TGF-
after 10 days
of treatment, this depended on the design of the experiment and slow
growth rate of tsFHI cells (cell counts were made every 5 days).
Inhibition of DNA synthesis (evaluated as incorporation of radioactive
thymidine into cellular DNA) was very strong (>78%) already after 5 days and was essentially complete by 7 days (data not shown). Also, in
a few experiments we have compared cells treated with TGF-
for 7 or
14 days, with essentially identical results. Taken together, these
findings indicate that growth arrest can be induced in tsFHI cells
without involving any of the CKIs they are known to express and provide
further evidence in support of the notion that inhibition of growth and
cell differentiation are separable phenomena in these cells. These
results are also of interest in view of the proposed role of TGF-
in
intestinal cell growth regulation in vivo (6, 37) and its possible role
in colon carcinogenesis (17).
Growth inhibition by culture of tsFHI cells in DMD at the permissive
temperature did, in contrast, result in moderate increases (2-fold)
in the cellular levels of p21 and cyclins
D2 and
D3 (Fig. 2) and in a slightly
increased association of Cdk2 with cyclin E (Fig.
5E). Although these changes appear
relatively minor, the presence of large amounts of cyclin
D3 in p21-containing complexes (Fig. 5A, lane 3) is intriguing. In
proliferating tsFHI cells, this cyclin was never observed in
CKI-containing complexes (Fig. 5A, lane
1), and it is tempting to speculate that inhibition
of cyclin D3-Cdk2/4 activity by
p21 may have been an important factor in cell behavior. Formation of
such complexes might also have had a stabilizing effect on cyclin
D3, explaining its increased cellular levels (Fig. 2A, lanes
3-6).
Induction of cell differentiation by a temperature shift had much more extensive effects on the cell cycle regulatory proteins we have studied than growth arrest alone. Most of these changes are summarized in Fig. 6 and are highlighted by the marked but temporally distinct increases in p21 and p27 expression (Figs. 1-3). The known characteristics of these two CKIs help in the interpretation of their possible functions and mechanisms of action.
p21 is a remarkable multifunctional protein: it can inhibit essentially all cyclin-Cdk complexes involved in pRb phosphorylation (28, 48); it can prevent cyclin's activation (phosphorylation) by cyclin-dependent kinase-activating kinases (CAK) (1); it inhibits the activity of cyclin E-Cdk2 complexes required for entry into the S phase of the cell cycle, even in pRb-deficient cells (25); and it interferes with DNA replication at two different steps, requiring the contribution of cyclin A-Cdk2 and PCNA, both inhibited by p21 (45). Paradoxically, at lower concentrations, p21 may actually stimulate the kinase activity of Cdks, promoting their association with cyclins (24). There is also strong evidence indicating that p21 may have positive (4, 18) or negative (7) effects on differentiation independently of its association with cyclins or Cdks.
p21 expression in tsFHI cells induced to differentiate had the following characteristics: 1) it increased rapidly, being noticeable as early as 8 h after the temperature shift; 2) it was apparently regulated at the transcriptional level, since approximately equivalent increases in protein and mRNA levels were observed; and 3) it was transient, its decline preceding the appearance of functional or morphological markers of differentiation (Fig. 2D) (37). This last observation, in particular, indicates that the primary role of p21 in tsFHI cells may have been in growth arrest, acting at the level of pRb phosphorylation. A rapid decline in the fraction of hyperphosphorylated pRb (Fig. 7A) and in the kinase activity associated with cyclin E-Cdk2 (Fig. 7D) was temporarily correlated with induction of p21 expression. Specific targets for p21 action could be inferred from analysis of the cyclins and Cdks found in p21 immunoprecipitates (Fig. 5) summarized in Fig. 6. On the basis of our data, three cyclin-Cdk complexes could be implicated in pRb phosphorylation: cyclin D1-Cdk4, cyclin D3-Cdk4, and cyclin E-Cdk2. Of these, only cyclin E-Cdk2 was found to display kinase activity (Fig. 7, C and D). The reasons for this unusual behavior of tsFHI cells may rest on the observation that Cdk2 associated with cyclin D immunoprecipitates was entirely in the inactive form (Fig. 5C) and Cdk6, although expressed by tsFHI cells (Fig. 2), was never observed in complexes with cyclin D or any of the other proteins examined. In p21 immunoprecipitates, induction of cell differentiation was accompanied by a marked increase in cyclin D3 (see above), a modest but significant increase in cyclin D1, and an essentially complete loss of active Cdk2 (likely associated with cyclin E). Thus p21 would have the potential to inhibit all cyclin-Cdk complexes likely to be involved in pRb phosphorylation in tsFHI cells. This scenario should, however, include p16, which may have played an important supporting role by sequestering a large fraction of cellular Cdk4, explaining the relatively small amounts detected in cyclin D immunoprecipitates. These considerations do not exclude the possibility that p21 may have also produced growth-inhibitory effects by interacting with other known targets, such as cyclin A-Cdk2 and PCNA, but the drastic reduction in cyclin A expression in differentiated cells indicates that such a function would likely be redundant. The decline in p21 expression during the second phase of cell differentiation may be interpreted to suggest that, having performed its growth-inhibitory functions, this CKI was no longer required. Immunofluorescence staining indicated, however, that all nuclei remained positive for p21, although at much reduced intensity, leaving open the alternative interpretation that such reduced levels were sufficient for continued inhibition of cell growth. An open question is whether the observed decline in p21 levels starting 2-3 days before induction of APN and DPPIV activities was a prerequisite for expression of differentiation markers, as observed in keratinocytes, where high levels of p21 were found to inhibit differentiation (7). Interestingly, the main conclusions of this study are also consistent with observations made on Caco-2 cells (9), where a G1/S block was demonstrated 3 days after confluency, preceding by 3 days the appearance of differentiated traits. In these cells, cell cycle block was also associated with suppression of Cdk2 and Cdk4 kinase activities and a concomitant increase in binding of p21 to Cdk2-containing complexes, leading the authors to conclude that inhibition of Cdk2 and Cdk4 was the main factor leading to G1 arrest, an event temporally dissociated from cell differentiation (9).
The most striking result from this study is the fact that p27 failed to complex with cyclins and Cdks, despite its fivefold increase in differentiating cells. This observation seems to exclude a significant role for this CKI in growth inhibition but opens the exciting possibility that it may have been directly involved in the induction of morphological and functional markers of differentiation by interacting with other intracellular regulators. The timing of p27 induction in cells shifted to 39°C correlated well with expression of APN and DPPIV, preceding it by 1-2 days. This conclusion is strengthened by our recent observation that forced overexpression of p27, in the normal human intestinal epithelial cell line HIEC6, by infection with a recombinant Adp27 adenovirus resulted in induction of cell differentiation (and expression of APN and DPPIV) 6 days after infection (unpublished observations). The absence of an SV40 T-Ag in such a model is further evidence that the main conclusions of this study were not influenced by this protein. It is also noteworthy that, in tsFHI cells shifted to 39°C, the increase in p27 protein was not accompanied by significant changes in its mRNA levels. A possible explanation for the latter finding is that a lower degree of p27 phosphorylation, due to reduced or nearly absent cyclin E-Cdk2 activity, may have been responsible for a greater stability and thus increased concentration of this protein, a mechanism of major importance in determining p27 levels in a number of cell lines (29). The conclusion that p27 is more directly related than p21 to expression of differentiated traits in intestinal epithelial cells is also consistent with the normal development of the intestinal tract in p21 knockout mice (8). In that study, the authors concluded that any essential role for p21 in terminal cell differentiation must be redundant (8). Our findings with tsFHI cells would indicate that p21 and p27 may play a role, although at different points in the differentiation process (growth arrest and induction of differentiated genes, respectively), suggesting that double (p21 and p27)-knockout animals might be much more informative.
Among all other cell cycle regulatory proteins investigated, D-type cyclins were of particular interest. The cyclin D1 gene is frequently overexpressed in human colon cancer cells and has been proposed to contribute to their abnormal growth and tumorigenicity (2). Overexpression of cyclin D1 and Cdk4 occurs in intestinal adenomas (50) and is associated with increased cell proliferation in premalignant neoplastic cells, with increased cyclin D1 immunoreactivity associated with more severe dysplasia (50). It is also noteworthy that this cyclin has been found to perform cellular functions not related to pRb phosphorylation. For example, cyclin D1 was found to potentiate transcription of estrogen receptor-regulated genes and to mediate this activation independent of complex formation with a Cdk partner (30). Similarly, cyclin D1, like B-Myb, was found to strongly activate the HSP-70 promoter via the heat shock element, again independently of pRb phosphorylation (23). It is therefore of interest that, even after several days in the differentiation process when no pRb phosphorylation could be detected, a constant level of cyclin D1 was maintained, and cyclins D2 and D3 were considerably induced. It is conceivable that high levels of D-type cyclins may be required for activation of differentiation-dependent intestinal genes.
Our working hypothesis concerning the role of CKIs in tsFHI cell growth
regulation and differentiation, highlighting the main results of this
study, is presented in Fig. 8. We
hypothesize that the first event is represented by induction of p21
expression through a p53-independent mechanism(s), without excluding a
complementary role for p53 and the SV0 T-Ag. High levels of p16,
inhibiting cyclin D-Cdk4 complexes, would make these cells primarily
dependent on cyclin E-Cdk2 for pRb phosphorylation; this would
represent the main target for p21-mediated inhibition of growth,
possibly also involving inhibition of CAK activity (resulting in
reduced Cdk2 phosphorylation; Fig.
5A). Other possible targets of p21, cyclin A-Cdk2 and PCNA, are less likely to play a role, but we have
observed a dramatic decline in cyclin A in differentiated cells.
Inhibition of cyclin E-Cdk2 kinase activity could result in reduced
phosphorylation and thus greater stability of p27, making it available
for interaction with unknown cellular mediator(s) of cell
differentiation, independently of cyclin-Cdk function.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-48331.
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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. Quaroni, Sect. of Physiology, T8 008A Vet Res Tower, Cornell University, Ithaca, NY 14853 (E-mail: aq10{at}cornell.edu).
Received 8 January 1999; accepted in final form 26 February 1999.
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