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Involvement of p21(WAF1/Cip1) and p27(Kip1) in intestinal epithelial cell differentiation

Jean Q. Tian and Andrea Quaroni

Section of Physiology, Cornell University, Ithaca, New York 14853


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta 1 (TGF-beta 1): 1) 31°C in GM (maximal cell growth), 2) 31°C in GM-1 ng/ml TGF-beta 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).

L929.06 cells, used as a positive control in the kinase assays, were obtained from Dr. E. Aubrey Thompson (Dept. of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX). They were cultured in DMEM supplemented with 10% FBS.

HeLa cells were infected with a recombinant adenovirus (Adp27, obtained from Dr. Prem Seth, Medicine Branch, National Cancer Institute, Bethesda, MD) to induce p27 overexpression and used as a control in the analysis of p27 complexes. Cells were infected with Adp27 at 100 or 500 plaque-forming units per cell. In the experiments described here they were used 6 days after infection. HeLa and 3T3 cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured in DMEM supplemented with 10% FBS.

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 [gamma -32P]ATP, and pRb-GST (Santa Cruz Biotechnology) at 1 µg/reaction or histone H1 (Boehringer Mannheim) at 2 µg/reaction. After 30 min, the reactions were stopped by addition of 30 µl of 2× SDS sample buffer. Radiolabeled substrates were separated from immunocomplexes by SDS-PAGE (12%). Gels were fixed, dried, and then exposed to X-ray film for autoradiography.

Cdk4- and Cdk6-associated kinase activities were determined exactly as described previously (35).

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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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-beta 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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Fig. 1.   Immunofluorescence staining of tsFHI cells for p21 (A, C, E, and G) and total nuclei with 4',6-diamidino-2-phenylindole (B, D, F, and H) or double stained with antibodies to p53 (I) and p21 (J). Cells were cultured in growth medium (GM) at 31°C (A, B, I, and J), in GM-1 ng/ml transforming growth factor (TGF)-beta 1 at 31°C (C and D), and in differentiation medium (DMD) at 39°C for 1 day (E and F) or in DMD at 39°C for 6 days (G and H). Arrows in I and J point to a nucleus intensely stained for p21 but only relatively weakly for p53.

Double labeling of growing cells for p53 and p21 also revealed that expression of these two proteins did not correlate: essentially all nuclei were stained, with variable intensity, for p53, and the few p21-positive nuclei often corresponded to lower levels of intensity of the p53 stain (Fig. 1, I and J, arrow). These results indicate that, in tsFHI cells, p21 expression may not be dependent on p53 stimulation and may correlate with cell differentiation rather than growth arrest.

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-beta 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).

Expression of cyclins, Cdks, and CKIs was evaluated at the protein level by Western blotting with use of total cell lysates (Fig. 2). Figure 2 is representative of the results obtained in four separate experiments. Overall, our main findings can be summarized as follows.


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Fig. 2.   Expression of cyclin-dependent kinase inhibitors (CKIs), E- and D-type cyclins, and cyclin-dependent kinases (Cdks) in tsFHI cells. Total cell lysates were prepared from tsFHI cells grown under 6 different culture conditions, separated by SDS-PAGE, then transferred onto nitrocellulose membranes, and finally immunoblotted with antibodies directed to indicated proteins. Lane 1, GM at 31°C for 1 day (GM); lane 2, GM containing 1 ng/ml TGF-beta 1 at 31°C for 1 day (TGF-beta ); lane 3, DMD at 39°C for 1 day (DMD); lane 4, DMD at 39°C for 1 day (DMD1); lane 5, DMD at 39°C for 3 days (DMD3); lane 6, DMD at 39° for 7 days (DMD7).

1) p15, p18, p19, and p57 could not be detected in tsFHI cells under any of the culture conditions used in this study (data not shown). The activity and specificity of the antibodies used for immunoblotting these proteins were confirmed with positive controls (HeLa and 3T3 cells) and specific blocking peptides obtained from Santa Cruz Biotechnology.

2) Both p21 and p27 were markedly elevated in tsFHI cells shifted to 39°C. The level of p21 protein after 24 h at 39°C was already about fivefold higher than that at 31°C in GM. However, after an early peak (maximum increase 6- to 8-fold at 1-2 days), p21 expression decreased gradually with prolonged incubation at 39°C. p21 protein was also induced slightly (1.4- to 1.7-fold) in tsFHI cells growth-arrested at 31°C in DMD. Growth arrest induced by TGF-beta 1 produced no significant changes in p21 levels.

3) The p27 expression pattern differed from p21 expression in two important respects: it was not affected by growth arrest at 31°C (in DMD or with TGF-beta 1), and its level continued to increase with prolonged incubation at 39°C, reaching a plateau (corresponding to >5-fold stimulation) by 7-9 days.

4) p16 was present in large, relatively constant amounts and increased only slightly in cells cultured in DMD at 31°C or after prolonged incubation (5-7 or more days) at 39°C.

5) Expression of cyclins D2 and D3 was markedly increased (4- to 8-fold) in tsFHI cells induced to differentiate; in contrast, cyclin D1 levels were relatively constant, declining by only 20-40% in cells in which growth was arrested in DMD at 31°C or during the first 3-5 days of incubation at 39°C. Cyclin E levels were always high, increasing by ~50% in growth-arrested and differentiated cells.

6) The protein levels of Cdk2 and Cdk4 were also relatively constant, increasing only slightly (<= 40%) after 3-5 days of incubation at 39°C. Cdk6 underwent a gradual, moderate increase (up to 70%) in differentiated cells.

To further characterize the kinetics of induction of p21, p27, and cyclins D2 and D3, their protein levels were evaluated at different times (from 0 to 48 h or from 0 to 15 days in separate experiments, each repeated 4 times) after the temperature switch (Fig. 3). Activities of DPPIV and APN, markers of tsFHI cell differentiation (37), were determined in parallel cultures (Fig. 3D).


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Fig. 3.   Induction of p21, p27, cyclins D2 and D3, dipeptidyl peptidase IV (DPPIV), and aminopeptidase N (APN) in tsFHI cells cultured at 39°C. Total cell lysates were prepared from tsFHI cells cultured continuously at 31°C in GM (optimal growth condition) or after a shift to 39°C in DMD for indicated times (4-48 h in A and B and 1-15 days in C and D), then transferred onto nitrocellulose membranes, and finally sequentially immunoblotted with antibodies directed to indicated proteins. Histograms show densitometric analysis of bands in corresponding blots. DPPIV and APN specific activities (D) were determined on total cell homogenates. * Significantly different from 31°C samples (P < 0.05).

Induction of p21 was rapid, starting at 8 h and reaching a peak by 36 h (Fig. 3A). After the peak, p21 expression returned (by 7-9 days) to a level comparable to that of cells grown in GM at the permissive temperature (Fig. 3C). In contrast, p27 induction was a slower, gradual process (Fig. 3A). Its induction peaked at ~9 days (Fig. 3C) and thereafter remained relatively constant for the duration of the experiment (15 days). A marked increase in both differentiation markers (Fig. 3D) followed by 1-2 days the increase in p27 levels.

Cyclins D2 and D3 displayed induction kinetics similar to those of p27 (Fig. 3B), reaching peak levels by 5-7 days (not shown).

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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Fig. 4.   A: RNase protection assay (RPA) of mRNAs in tsFHI cells cultured under 6 different conditions: 31°C in GM (GM), 31°C in GM-1 ng/ml TGF-beta 1 (TGF-beta ), 31°C in DMD (DMD), or 39°C in DMD for 1 day (DMD1), 3 days (DMD3), and 7 days (DMD7). B: corresponding densitometric analysis. In all cases, total cellular RNA was isolated, hybridized with 3 different mixtures of antisense riboprobes produced using a Multi-RiboQuant RPA kit from Pharminigen, and then digested with RNase (H + T1). Protected fragments were separated on a 6% precast sequencing gel (Stratagene) and finally exposed to X-ray film. L32 and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) represent reference "housekeeping" genes added to verify equal loads of RNA.

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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Fig. 5.   Analysis of protein complexes formed by CKIs, Cdks, and cyclins in tsFHI cells cultured under 6 different conditions: 31°C in GM (GM), 31°C in GM-1 ng/ml TGF-beta 1 (TGFbeta ), 31°C in DMD (DMD), or 39°C in DMD for 1 day (DMD1), 3 days (DMD3), and 7 days (DMD7). B: immunoblots obtained with anti-p27 immunoprecipitates from HeLa cells infected with a recombinant Adp27 adenovirus at 100 (lane 1) or 500 (lane 2) plaque-forming units/cell. Equal amounts of total cell lysates were separately immunoprecipitated with antibodies directed against p21 (IP: alpha -p21), p27 (IP: alpha -p27), p16 (IP: alpha -p16), cyclin D (D1, D2, and D3, IP: alpha -cyclin D), or cyclin E (IP: alpha -cyclin E). In all cases, immunoprecipitates were then separated by SDS-PAGE, blotted onto nitrocellulose, and finally sequentially stained with antibodies specific for cyclins A, E, D1, D2, and D3, and Cdk2, Cdk4, and Cdk6. Only selected results are presented.

1) Cyclin D2 and Cdk6 could not be detected in any of the immunoprecipitates (not shown).

2) Cyclin A, cyclin E, cyclin D1, cyclin D3, Cdk2, and Cdk4 were present in p21 immunoprecipitates (Fig. 5A, lanes 1-6). The amount of cyclin A associated with p21 decreased rapidly in cells cultured at 39°C, concomitant with a marked increase in cyclin D1 and, particularly, cyclin D3 (Fig. 5A). It is noteworthy that cyclin D3 was undetectable in immunoprecipitates obtained from cells cultured under optimal growth conditions (at 31°C in GM) and at a very low level in cells in which growth was arrested with TGF-beta 1 (Fig. 5A, lanes 1 and 2). Figure 5A also shows that both known forms of Cdk2, i.e., phosphorylated kinase-active (lower band) and unphosphorylated kinase-inactive (upper band), were associated with p21. Interestingly, only the lower (kinase-active) band showed a marked decline on differentiation at 39°C (Fig. 5A). The amount of Cdk4 associated with p21 increased slightly with the temperature shift and in the cells cultured in DMD at 31°C (Fig. 5A), but it is important to point out that, in direct comparisons, three- to fourfold higher amounts of Cdk4 were found in p16 immunoprecipitates (Fig. 5C).

3) p16 immunoprecipitates contained only Cdk4; no Cdk2 (Fig. 5C), Cdk6, or any of the other cyclins tested (not shown) were found.

4) p27 immunoprecipitates contained no Cdk4 or Cdk6; only very small amounts of Cdk2 and cyclins A, D1, and E could be detected (Fig. 5A, lanes 7-12). This striking result was not due to insufficient p27 antibody, since it did not change with use of two- to threefold higher amounts of antibody for immunoprecipitation. In direct comparisons (immunoprecipitates obtained from equal aliquots of the same cell lysates), the levels of cyclins and Cdks associated with p27 were clearly much lower than those associated with p21 (Fig. 5A). To confirm that this result was not due to inability of the p27 antibody to immunoprecipitate protein complexes, we have used as positive control HeLa cells induced to overexpress p27 by infection with a recombinant adenovirus carrying the p27 gene at two different concentrations (100 or 500 plaque-forming units/cell). As shown in Fig. 5B, cyclins A, E, D1, and D3 and Cdk2 and Cdk4 could be detected in p27 immunoprecipitates obtained from such cells.

To further dissect the complex association among Cdk2, Cdk4, cyclin E, cyclin D, and p21, we immunoprecipitated cyclin D (the different forms were immunoprecipitated together, as illustrated in Fig. 5D, or individually) or cyclin E from the cell lysates and analyzed the other proteins present. Only the relevant information from these studies is illustrated in Fig. 5. Cdk4 was only detected in the cyclin D immunoprecipitates (Fig. 5D), which also contained the kinase-inactive slower-migrating form of Cdk2 but none of the active form (Fig. 5D). The amounts of Cdk2 and especially Cdk4 associated with cyclin D increased with tsFHI cell differentiation at 39°C (Fig. 5D). In cyclin E immunoprecipitates, we detected both forms of Cdk2, but no Cdk4 (Fig. 5E). It is important to note that Cdk6 could not be detected in any of these immunoprecipitates (data not shown).

Overall, our results, summarized in Fig. 6, indicate that the main G1/S cyclin-Cdk complexes present in tsFHI cells are 1) cyclin D associated with inactive Cdk2 or, more so in differentiated cells, Cdk4, 2) cyclin E-Cdk2, and 3) cyclin A-Cdk2 (not shown). With respect to CKIs, 1) p16 only associated with Cdk4; 2) p21 associated with cyclins D1/D3-Cdk2 or cyclins D1/D3-Cdk4, cyclin A-Cdk2, and cyclin E-Cdk2. With differentiation, p21 dissociated from cyclin A-Cdk2 complexes (cyclin A levels were markedly reduced); in p21-containing complexes there was also a marked increase in cyclins D1 and D3 and a decrease in active Cdk2; and 3) p27 formed only very small amounts of complexes with cyclin D1-Cdk4, cyclin E-Cdk2, and cyclin A-Cdk2. This surprising finding suggests that most of the p27 induced in tsFHI cells at 39°C remained free or associated with different unknown cellular components.


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Fig. 6.   Summary of main findings of this study: main cyclin-Cdk-CKI complexes found in tsFHI cells and relative changes in distribution of these proteins or abundance of complexes observed with growth arrest (cells cultured at permissive temperature in DMD or with TGF-beta 1) or differentiation (cells cultured at 39°C in DMD).

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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Fig. 7.   A and B: expression of retinoblastoma protein (pRb), SV40 T-antigen (T-Ag), and p53 in tsFHI cells. Total cell lysates were prepared from tsFHI cells cultured at 31°C in GM (lane 1) or switched to DMD at 39°C for indicated time periods, subjected to SDS-PAGE, then transferred onto nitrocellulose membranes, and finally immunoblotted with antibodies directed to indicated proteins. Hyperphosphorylayed form of pRb appears as a lower-mobility band, disappearing after 16-24 h of incubation at nonpermissive temperature. C: Cdk2, Cdk4, Cdk6, and cyclin D-associated kinase activities in tsFHI cells cultured at 31°C in GM and in control L929.06 cells. In the case of Cdk4, 2 different antibodies were used for immunoprecipitation: lanes 3 and 10, alpha -Cdk4 (sc-260) from Santa Cruz; lanes 4 and 11, alpha -Cdk4 (06-139) from Upstate Biotechnology; lanes 5 and 12 and lanes 6 and 13, immunoprecipitates obtained with 2 different amounts (2 and 4 mg, respectively) of the same alpha -Cdk6 (sc-177) antibody from Santa Cruz; lanes 7 and 14, immunoprecipitates that contain all 3 cyclin D forms (D1, D2, and D3). No primary antibody was added to negative (beads) controls (lanes 1 and 8). D: kinase activity in cyclin E and Cdk2 immunoprecipitates from tsFHI cells cultured at 31°C in GM (lanes 2 and 9) or shifted to 39°C in DMD (lanes 3-7 and 10-12) for indicated periods of time. Immunoprecipitation was performed with cyclin E or Cdk2 antibodies from equal amounts of total cell lysates. No primary antibody was added to negative (beads) controls (lanes 1 and 8). In C and D, pRb was used as a substrate, but similar results were obtained with histone H1 as a substrate.

It is generally recognized that cyclin D-associated kinases are primarily responsible for pRb phosphorylation in G1, but cyclin E-Cdk2 complexes may also play an important role. There is also strong evidence to suggest that the kinase activity of cyclin E-Cdk2 is essential for entry into the S phase of the cell cycle even in cells lacking pRb (25). We have therefore evaluated these different kinase activities in tsFHI cells. Surprisingly, only Cdk2 was found to be active in cells cultured under optimal growth conditions (Fig. 7C), growth arrested, or induced to differentiate (data not shown). No kinase activity was detected in immunoprecipitates obtained with a mixture of anti-cyclin D antibodies, including antibodies recognizing D1, D2, and D3 forms (Fig. 7C, lane 7). All three kinases could be detected in control L929 cells, which also displayed comparatively higher levels of Cdk2-associated kinase activity.

To determine whether the cyclin E-Cdk2 kinase activity was altered with tsFHI cell differentiation, we separately immunoprecipitated complexes with antibodies directed against Cdk2 or cyclin E from total cell lysates and then measured their associated kinase activities. Histone H1 (not shown) and pRb (Fig. 7D) were phosphorylated by cyclin E-Cdk2 complexes obtained from cells cultured under optimal growth conditions (Fig. 7D, lanes 2 and 9). The kinase activity associated with cyclin E-Cdk2 complexes declined rapidly in cells induced to differentiate at 39°C (Fig. 7D, lanes 3-7 and 10-12), and after 3 days at the nonpermissive temperature, band density was not significantly different from negative controls consisting of beads alone (without specific antibody; Fig. 7D, lanes 1 and 8).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta 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-beta 1. This is surprising, because previous reports have implicated p15 (19) and p27 (39) as key mediators of TGF-beta 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-beta . p16 and p21 were also unaltered in their cellular levels or association with Cdks and cyclins in TGF-beta -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-beta 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-beta 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-beta 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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Fig. 8.   Role of CKIs in tsFHI cells: our working hypothesis concerning function of p16, p21, and p27 in growth regulation and differentiation of intestinal epithelial cells. CAK, cyclin-dependent kinase-activating kinase; PCNA, proliferating cell nuclear antigen; RPC, replication protein C.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-48331.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 276(6):C1245-C1258
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