Department of Surgery, University of Texas Medical Branch, Galveston, Texas 77555
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ABSTRACT |
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The cellular mechanisms regulating intestinal proliferation and differentiation remain largely undefined. Previously, we showed an early induction of the cyclin-dependent kinase (CDK) inhibitor p21Waf1/Cip1 in Caco-2 cells, a human colon cancer line that spontaneously differentiates into a small bowel phenotype. The purpose of our present study was to assess the timing of cell cycle arrest in relation to differentiation in Caco-2 cells and to examine the mechanisms responsible for CDK inactivation. Caco-2 cells undergo a relative G1/S block and cease to proliferate at day 3 postconfluency; an increase in the activity of terminally differentiated brush-border enzymes (sucrase and alkaline phosphatase) was noted at day 6 postconfluency. Cell cycle block was associated with suppression of both CDK2 and CDK4 activities, which are important for G1/S progression. Treatment of the CDK immune complexes with the detergent deoxycholate (DOC) resulted in restoration of CDK2, but not CDK4, activity at day 3 postconfluency, suggesting the presence of inhibitory protein(s) binding to the cyclin/CDK2 complex at this time point. An increased binding of p21Waf1/Cip1 to CDK2 complexes at day 3 postconfluency was noted, suggesting a potential role for p21Waf1/Cip1 in CDK2 inactivation; however, immunodepletion of p21Waf1/Cip1 from Caco-2 protein extracts demonstrated that p21Waf1/Cip1 is only partially responsible for CDK2 suppression at day 3 postconfluency. A decrease in the cyclin E/CDK2 complex appears to contribute to the CDK2 inactivation noted at days 6 and 12 postconfluency. Taken together, our results suggest that multiple mechanisms contribute to CDK suppression during Caco-2 cell differentiation. Inhibition of CDK2 and CDK4 leads to G1 arrest and inhibition of proliferation that precede Caco-2 cell differentiation.
gut differentiation; cyclin-dependent kinases; cell cycle; cyclin-dependent kinase inhibitor
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INTRODUCTION |
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THE EPITHELIUM OF THE gastrointestinal tract is a complex and dynamic tissue composed of numerous cell types with important cellular functions, including digestion, absorption, barrier and immune function, and peptide secretion (35). The mammalian intestinal mucosa undergoes a process of continual renewal characterized by active proliferation of stem cells localized near the base of the crypts, progression of these cells up the crypt-villus axis with cessation of proliferation, and subsequent differentiation into one of the four primary cell types (i.e., absorptive enterocytes, goblet cells, Paneth cells, and enteroendocrine cells) (7, 16, 36, 38, 43). The differentiated enterocytes, which make up the majority of the cells of the gut mucosa, then undergo a process of programmed cell death (i.e., apoptosis) and extrusion into the gut lumen (22, 37). Remarkably, this entire process of proliferation, differentiation, and apoptosis and extrusion occurs over a 3- to 5-day period, depending on the species (7, 16, 36-38, 43). The cellular mechanisms regulating this tightly regimented process have not been clearly defined; however, this topic represents an area of active investigation, since the delineation of this process will lead to a better understanding of normal gut mucosal growth.
The mammalian cell cycle is regulated by the sequential activation and inactivation of a highly conserved family of cyclin-dependent kinases (CDKs) (31, 46). CDK activation requires the binding of a regulatory protein (i.e., cyclin) (24) and is controlled by both positive and negative phosphorylation (29). Cell cycle progression is regulated at two key checkpoints, the G1/S and the G2/M transition points (32, 33). Progression through early to mid-G1 is dependent on CDK4, and possibly CDK6, which are activated by association with one of the three D-type cyclins (D1, D2, and D3) (40). Progression through late G1 and into the S phase requires activation of CDK2, which is sequentially regulated by cyclins E and A, respectively (11, 19). The activities of the CDKs can be inhibited by the binding of CDK inhibitory proteins (13, 41). Two families of CDK inhibitory proteins have been identified. The first family consists of p15Ink4b, p16Ink4a, p18Ink4c, and p19Ink4d, which appear to selectively bind and inhibit CDK4 and CDK6 (17, 18, 21, 39), whereas members of the second family, consisting of p21Waf1/Cip1, p27Kip1/Pic2, and p57Kip2, are universal inhibitors of the cyclin/CDK complexes (13, 41). In addition, important targets of CDK4 and CDK2 include the retinoblastoma protein (pRb) and the pRb-related proteins p107 and p130 (26, 47). In their hypophosphorylated form, the pRb family of proteins can sequester and inactivate the E2F family of transcription factors that regulate genes that participate in S phase entry.
The human colon cancer cell line Caco-2 spontaneously differentiates to a small bowel-like phenotype, as indicated by dome formation, presence of microvilli, and expression of brush-border enzymes (i.e., sucrase and alkaline phosphatase) after confluency. Caco-2 has served as a useful in vitro model to further delineate mechanisms triggering the terminal differentiation process in enterocytes (27, 34, 50). Previously, we showed an early induction of the CDK inhibitor p21Waf1/Cip1 occurring in Caco-2 cells on day 3 postconfluency that precedes induction of sucrase-isomaltase gene expression (14). Because p21Waf1/Cip1 is a universal inhibitor of CDKs, its induction during Caco-2 differentiation may be sufficient to inhibit CDK activity and induce cell cycle arrest. Therefore, the purpose of our present study was to assess the timing of cell cycle arrest in relation to differentiation of the Caco-2 cell line. In addition, we evaluated the role of p21Waf1/Cip1 in inhibiting CDK2 activity during Caco-2 differentiation.
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MATERIALS AND METHODS |
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Materials. Radioactive compounds were purchased from DuPont-NEN (Boston, MA). Immobilon P nylon membranes for Western blots were purchased from Millipore (Bedford, MA), and X-ray film was from Eastman Kodak (Rochester, NY). The human glutathione S-transferase retinoblastoma protein (GST-Rb) and all antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). The enhanced chemiluminescence (ECL) system for Western immunoblot analysis and the cell proliferation kit were purchased from Amersham (Arlington Heights, IL). Concentrated protein assay dye reagent was purchased from Bio-Rad Laboratories (Hercules, CA). Tissue culture media and reagents were obtained from GIBCO BRL (Grand Island, NY). All other reagents were of molecular biology grade and were purchased from either Sigma (St. Louis, MO) or Amresco (Solon, OH).
Cell culture, cell counting, and 5-bromo-2'deoxyuridine immunohistochemistry. The human colon cancer cell line Caco-2, obtained from the American Type Culture Collection (Manassas, VA), was maintained in modified Eagle's medium supplemented with 15% (vol/vol) FCS. The cells were seeded at 1 × 106 cells in 25-cm2 flasks and maintained in a humidified atmosphere of 95% air-5% CO2 at 37°C. Studies were performed on cells at various times either before confluency was reached (preconfluency) or postconfluency. For assessment of cell proliferation, Caco-2 cells were counted over a time course using a hemocytometer. 5-Bromo-2'deoxyuridine (BrDU) immunohistochemistry was performed on preconfluent and day 3 postconfluent cells using the cell proliferation kit as described by the manufacturer, after a 60-min pulse with BrDU at 37°C.
Enzyme assays. Caco-2 cells were harvested with trypsin and washed in PBS. The cell pellet was homogenized and sonicated in Tris-mannitol buffer [2 mM Tris and 50 mM mannitol (pH 7.1)] at 4°C. Sucrase (EC 3.3.1.48) activity was measured according to the method of Messer and Dahlqvist (28). Alkaline phosphatase (EC 3.1.3.1) activity was determined using an alkaline phosphatase determination kit from Sigma. Values are expressed as milliunits per milligram protein; one unit is defined as the activity that hydrolyzes 1 µmol substrate/min at 37°C. Proteins were assayed by the method of Bradford (4).
Flow cytometry. Caco-2 cells were harvested with trypsin at various time points, washed twice with PBS, and then resuspended in PBS. Cells were then fixed in 80% ethanol for 30 min at room temperature and stored at 4°C. Before processing, cells were collected by centrifugation and stained by addition of 1 ml of propidium iodine solution (50 µg/ml). RNase A (100 µg/ml) was added, and the sample was incubated for 15 min at room temperature. Cell cycle analysis was performed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA), and cell cycle distribution was analyzed by the Modfit LT program (Verity, ME).
Protein preparation, Western immunoblot, and immunoprecipitation. Caco-2 cells were lysed with TNN buffer [in mM: 50 Tris · HCl (pH 7.5), 150 NaCl, 0.5 Nonidet P-40, 50 NaF, 1 sodium orthovanadate, 1 dithiothreitol (DTT), and 1 phenylmethylsulfonyl fluoride, and 25 µg/ml each of aprotinin, leupeptin, and pepstatin A] at 4°C for 30 min. Lysates were clarified by centrifugation (10,000 g for 30 min at 4°C), and protein concentrations were determined using the method of Bradford (4). Western immunoblot analyses were performed as described previously (14). Briefly, protein samples (60 µg) were resolved by SDS-PAGE and then electroblotted to Immobilon P nylon membranes. Filters were incubated overnight at 4°C in blocking solution (Tris-buffered saline containing 5% nonfat dried milk and 0.05% Tween 20), followed by 3 h of incubation with the primary antibody. Filters were incubated with a horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG as a secondary antibody for 1 h. After four final washes, the immune complexes were visualized using ECL detection. For determination of p21Waf1/Cip1 bound to CDK2, lysates were immunoprecipitated with anti-CDK2 antibody, resolved by SDS-PAGE, and transferred to nylon membranes. The membranes were then probed with an antibody against p21Waf1/Cip1 (sc-397, Santa Cruz Biotechnology). Signals on the blots were visualized by autoradiography and quantitated by densitometry using a Lynx 5000 digital image analysis system.
Kinase assays. Caco-2 cells (preconfluent, confluent, and postconfluent) were lysed with TNN buffer, and protein samples (300 µg) were incubated with 1.5 µg of anti-CDK2 or anti-CDK4 antibodies. Immune complexes were recovered with protein A-Sepharose beads, washed twice with TNN buffer and once with kinase buffer [in mM: 25 HEPES (pH 7.4), 10 MgCl2, and 1 EGTA]. Pellets were resuspended in 40 µl of kinase buffer containing either 5 µg of histone H1 (Sigma; to measure CDK2-associated kinase activity) or GST-Rb (to measure CDK4-associated kinase activity) at 30°C for 30 min. The kinase reaction was terminated by addition of SDS sample loading buffer [50 mM Tris (pH 6.8), 100 mM DTT, 2% SDS, 0.1% bromphenol blue, and 10% glycerol]. The samples were then heated to 95°C for 5 min and resolved by 10% SDS-PAGE. The gels were dried, and the phosphorylated proteins were visualized by autoradiography and quantitated by densitometry.
DOC activation assays. Caco-2 cell extracts were immunoprecipitated with either anti-CDK2 or anti-CDK4 antibodies, and the resultant immune complexes were incubated with either 20 mM HEPES (pH 7.4) alone or 20 mM HEPES containing 0.8% DOC on ice for 20 min. DOC was removed by washing three times with TNN buffer and once with kinase buffer. CDK2 and CDK4 activities were then analyzed as described above.
Immunodepletion kinase assays. Protein extract (100 µg) from day 3 postconfluent Caco-2 cells was heated for 5 min at 95°C and then immunoprecipitated with IgG or antip21Waf1/Cip1 antibody (2.0 µg). The immunodepleted supernatant was mixed with protein extract (100 µg) from confluent (day 0) Caco-2 cells, incubated at 30°C for 30 min, and then immunoprecipitated with anti-CDK2 antibody. The associated kinase activity was assayed as described above.
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RESULTS |
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Association of Caco-2 cell differentiation with
G1 cell cycle arrest and cessation of
proliferation.
Previously, we showed that enterocytic differentiation of the Caco-2
cell line, which occurs after confluency, was preceded by induction of
the universal CDK inhibitor,
p21Waf1/Cip1, suggesting that
Caco-2 cell differentiation was associated with cell cycle arrest (14).
To further ascertain the cell cycle distribution of preconfluent
(day
2), confluent
(day
0), and postconfluent Caco-2 cells,
DNA flow cytometry was performed (Fig. 1A).
Our results indicate that 58% of preconfluent Caco-2 cells were in the
G0/G1
phase and 28% of the cells were in the S phase of the cell cycle. In
contrast, 73% of Caco-2 cells at day
3 postconfluency were in
G0/G1,
with only 19% in S phase; these percentages remained relatively stable
up to day
12 postconfluency. Therefore, our results demonstrate a 32% decrease in the S phase population, consistent with an induction of a relative
G1/S cell cycle block occurring in
day 3 postconfluent Caco-2 cells.
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Caco-2 cell differentiation is associated with decreases in CDK2 and CDK4 activities. To further examine the mechanisms responsible for G1 arrest at day 3 postconfluency and subsequent differentiation, we first determined CDK2 and CDK4 kinase activities in preconfluent and postconfluent Caco-2 cells. These CDKs are required for the progression from G1 to the S phase (31, 46) and can be inhibited by p21Waf1/Cip1. Caco-2 cell extracts from preconfluent, confluent (day 0), and postconfluent cultures were immunoprecipitated with either anti-CDK2 or anti-CDK4 antibodies, and the immune complexes were assayed for kinase activity using histone H1 (Fig. 2A) or a purified GST-Rb fusion protein (Fig. 2B) as substrates, respectively. As demonstrated by the densitometric analyses (Fig. 2, A and B, bottom), day 3 postconfluent cells had a 50% decrease in both CDK2 and CDK4 activities compared with preconfluent cells. A further decrease of CDK2 activity to almost undetectable levels was noted at days 6 and 12 postconfluency. The suppression of both CDK2 and CDK4 activities, occurring on day 3 postconfluency, coincides with a decrease in the S phase population (Fig. 1A). Potential mechanisms for the downregulation of CDK activities include the increased expression of one or more cell cycle inhibitor proteins present in postconfluent (day 3) Caco-2 cells or, alternatively, a decrease in cyclin/CDK complex formation.
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Suppression of CDK2 activity in day 3 postconfluent Caco-2 cells is associated with inhibitory protein(s) that bind CDK2. To assess the possibility of inhibitory protein(s) binding to the cyclin/CDK2 and/or cyclin/CDK4 complexes in differentiating Caco-2 cells, we used the detergent DOC, which has been shown to preferentially dissociate certain protein-protein interactions and leave the cyclin/CDK complex intact (48). The CDK2 immune complexes were treated with either buffer alone or buffer containing DOC for 20 min and then assayed for CDK2 activity (Fig. 3A). Treatment with DOC, but not with buffer alone, restored the kinase activity at day 3 postconfluency to preconfluent levels. However, CDK2 activity was not altered by DOC on days 6 and 12 postconfluency. This result indicates that one or possibly more inhibitory proteins contribute to the decrease of CDK2 activity in the day 3 postconfluent Caco-2 cells, but not in the days 6 and 12 postconfluent cells. In contrast, no increase of CDK4 activity was noted after treating the CDK4 immune complexes with DOC (Fig. 3B), which suggests that CDK4 inhibition in differentiating Caco-2 cells is not the result of the binding of inhibitory proteins.
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Potential role of p21Waf1/Cip1 in the suppression of CDK2 activity. Previously, we showed that the CDK inhibitor p21Waf1/Cip1 is induced in differentiating Caco-2 cells beginning on day 3 postconfluency (14). We next assessed the potential role of this protein in blocking CDK2 activity on day 3 postconfluency. p21Waf1/Cip1 inhibits the cyclin/CDK complex by binding the heterodimeric complex. To determine whether p21Waf1/Cip1 is bound to the cyclin/CDK2 complexes during Caco-2 differentiation, immunoprecipitation of the Caco-2 protein extracts was performed using anti-CDK2 antibody, and the immune complexes were assayed for the abundance of p21Waf1/Cip1, as shown in Fig. 5A. An increase in the amount of p21Waf1/Cip1 bound to CDK2 was noted at day 3 postconfluency and persisted on days 6 and 12 postconfluency.
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DISCUSSION |
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As intestinal epithelial cells progress up the crypt-villus unit, they enter a terminal differentiation program that in the absorptive enterocyte involves cessation of proliferation and expression of the terminally differentiated enzymes sucrase and alkaline phosphatase (7, 16, 36, 38, 43). In our present study, we demonstrate growth inhibition and a relative G1/S block of the gut-derived Caco-2 cell line at day 3 postconfluency that precede the increases of both sucrase and alkaline phosphatase activity. This is, to our knowledge, the first demonstration of G1/S block preceding Caco-2 cell differentiation. Others have also demonstrated the importance of G1 arrest in the subsequent differentiation of nonintestine-derived cell types. For example, Decker (8) demonstrated that nerve growth factor (NGF)-mediated differentiation of the rat pheochromocytoma cell line PC-12 to a neuronal phenotype is associated with G1 arrest and p21Waf1/Cip1 induction. Furthermore, cell cycle arrest was also noted during myoblast differentiation, interleukin-6-induced B cell differentiation, and differentiation of the NB4 promyelocytic cell line mediated by retinoic acid (2, 3, 30). Taken together, these results suggest that cell cycle arrest may be a prerequisite for terminal differentiation of various cell types.
Important regulators of the mammalian G1/S transition include the CDK2 and CDK4 proteins (31, 46). Suppression of CDK2 and CDK4 activities induces neuronal differentiation of mouse neuroblastoma cells (25); inhibition of CDK2 and CDK4 is associated with glial cell differentiation of central glia-4 cells and hexamethylene bisacetamide-induced differentiation of murine erythroleukemia cells, respectively (23, 42). Conversely, the overexpression of CDK2 can block NGF-induced differentiation of PC-12 cells (10). Concomitant with the increase in the G1 phase population of Caco-2 cells noted at day 3 postconfluency, we found a decrease in the kinase activities of both CDK2 and CDK4, suggesting that suppression of CDKs may be important in subsequent intestinal cell differentiation. This hypothesis is further supported by the recent in vivo findings of Chandrasekaran et al. (6) demonstrating a progressive decrease of CDK2 protein levels as cells progress up the crypt-villus axis, with undetectable levels noted in the villus.
CDK activity can be inhibited by multiple mechanisms, including decreased expression of cyclin or CDK proteins, decreased formation of cyclin/CDK complexes, negative phosphorylation, or increased binding of inhibitory protein(s). Several of these mechanisms appear to be responsible for suppression of CDK2 and CDK4 in differentiating Caco-2 cells. Treatment of the CDK2 immune complexes with the detergent DOC reversed the CDK2 suppression at day 3 postconfluency, but not at days 6 and 12, suggesting that CDK2 inactivation on day 3 is the result of binding of inhibitory protein(s). These findings were further supported by the fact that levels of cyclin E/CDK2 complexes were not decreased on day 3 postconfluency. Other mechanisms likely contribute to the sustained CDK2 inhibition noted on days 6 and 12 postconfluency. Previously, we showed a decrease in cyclin E and CDK2 expression in days 6 and 12 postconfluent cells. In the present study, we also noted a dramatic decrease in cyclin E/CDK2 complexes at these time points; therefore, the decreases noted in the cyclin E/CDK2 complexes may contribute to CDK2 suppression at days 6 and 12. In contrast to CDK2, CDK4 activity remains suppressed even after the addition of DOC. These results, in conjunction with our previous findings of decreased D-type cyclin expression (14), suggest that suppression of CDK4 is secondary to a decrease in D-type cyclin protein levels. Interestingly, levels of cyclin D1 fall rapidly in vivo in association with enterocytic differentiation (6), which further supports a role for the D-type cyclins in intestinal cell differentiation. Therefore, distinct mechanisms may be responsible for the suppression of the CDKs, depending on the specific CDK and the time point during the differentiation process.
Because the suppression of CDKs at day 3 postconfluency appears to be an important event in subsequent Caco-2 cell differentiation, we wanted to determine the potential inhibitory protein(s) responsible for CDK2 inactivation. Previously, we demonstrated an induction of p21Waf1/Cip1 protein in day 3 postconfluent Caco-2 cells (14); this early induction of p21Waf1/Cip1 in Caco-2 cells has been confirmed by Gartel et al. (15) and Abraham et al. (1). Therefore, we tested the hypothesis that p21Waf1/Cip1 is responsible for the inhibition of CDK2 activity noted on day 3 postconfluency. Levels of p21Waf1/Cip1 bound to CDK2 were increased by day 3 postconfluency; however, heated protein extracts from day 3 postconfluent Caco-2 cells depleted of p21Waf1/Cip1 were still able to partially inhibit CDK2 activity, although not as effectively as heated extracts not depleted of p21Waf1/Cip1. Our findings suggest that although p21Waf1/Cip1 appears to contribute to CDK2 inactivation on day 3 postconfluency, other inhibitory proteins are also likely to be involved in this process. Moreover, these results also suggest that additional mechanisms, other than p21Waf1/Cip1 induction, may contribute to the cell cycle arrest and subsequent differentiation of the Caco-2 intestinal cell line. This supposition is further supported by the in vivo findings of Brugarolas et al. (5) and Deng et al. (9), who independently demonstrated normal intestinal development in mice lacking p21Waf1/Cip1, suggesting either that p21Waf1/Cip1 is not essential for gut differentiation or, alternatively, that other mechanisms can compensate for its loss. In addition to p21Waf1/Cip1, the CDK inhibitors p27Kip1/Pic2 and p57Kip2 have been postulated to play a role in cellular differentiation (12, 13, 24, 49); however, we did not detect binding of either of these proteins to CDK2 in the day 3 postconfluent Caco-2 cells. Future studies will attempt to identify other CDK2 inhibitory protein(s) expressed during Caco-2 cell differentiation.
In conclusion, we have demonstrated that a relative G1/S cell cycle block and suppression of the CDK2 and CDK4 activities precede the increased differentiation of Caco-2 cells. We have also shown that multiple mechanisms are responsible for suppression of CDK activities observed during Caco-2 cell differentiation. These potential mechanisms include the downregulation of cyclin and CDK protein expression, inhibition of cyclin/CDK complex formation, the induction of the CDK inhibitor p21Waf1/Cip1, and the potential involvement of other inhibitory proteins. Identification of the cell cycle mechanisms regulating Caco-2 cell differentiation will provide important insights into the regulation of mammalian intestinal cell proliferation, differentiation, and subsequent apoptosis.
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ACKNOWLEDGEMENTS |
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We thank Drs. Mark R. Hellmich, Zizheng Dong, and E. Aubrey Thompson for helpful advice and discussion. In addition, we thank Eileen Figueroa and Karen Martin for manuscript preparation.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants RO1-DK-48498 and RO1-AG-10885 (to B. M. Evers), KO8-CA-64191 (to T. C. Ko), and PO1-DK-35608 and by the James E. Thompson Memorial Foundation.
Q.-M. Ding is a visiting scientist from the Institute of Radiation Medicine, Beijing, China.
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: B. M. Evers, Dept. of Surgery, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555.
Received 28 April 1998; accepted in final form 22 July 1998.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abraham, C.,
B. Scaglione-Sewell,
S. F. Skarosi,
W. Qin,
M. Bissonnette,
and
T. A. Brasitus.
Protein kinase C modulates growth and differentiation in Caco-2 cells.
Gastroenterology
114:
503-509,
1998[Medline].
2.
Andres, V.,
and
K. Walsh.
Myogenin expression, cell cycle withdrawal, and phenotypic differentiation are temporally separable events that precede cell fusion upon myogenesis.
J. Cell Biol.
132:
657-666,
1996[Abstract].
3.
Bocchia, M.,
Q. Xu,
U. Wesley,
Y. Xu,
T. Korontsvit,
F. Loganzo,
A. P. Albino,
and
D. A. Scheinberg.
Modulation of p53, WAF1/p21 and BCL-2 expression during retinoic acid-induced differentiation of NB4 promyelocytic cells.
Leuk. Res.
21:
439-447,
1997[Medline].
4.
Bradford, M. M.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
72:
248-254,
1976[Medline].
5.
Brugarolas, J.,
C. Chadrasekaran,
J. Gordon,
D. Beach,
T. Jacks,
and
G. Hannon.
Radiation-induced cell cycle arrest compromised by p21 deficiency.
Nature
377:
552-557,
1995[Medline].
6.
Chandrasekaran, C.,
C. M. Coopersmith,
and
J. I. Gordon.
Use of normal and transgenic mice to examine the relationship between terminal differentiation of intestinal epithelial cells and accumulation of their cell cycle regulators.
J. Biol. Chem.
271:
28414-28421,
1996
7.
Cheng, H.,
and
C. P. Leblond.
Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. V. Unitarian theory of the origin of the four epithelial cell types.
Am. J. Anat.
141:
537-561,
1974[Medline].
8.
Decker, S. J.
Nerve growth factor-induced growth arrest and induction of p21Cip1/WAF1 in NIH-3T3 cells expressing TrkA.
J. Biol. Chem.
270:
30841-30844,
1995
9.
Deng, C.,
P. Zhang,
J. W. Harper,
S. J. Elledge,
and
P. Leder.
Mice lacking p21CIP1/WAF1 undergo normal development, but are defective in G1 checkpoint control.
Cell
82:
675-684,
1995[Medline].
10.
Dobashi, Y.,
T. Kudoh,
A. Matsumine,
K. Toyoshima,
and
T. Akiyama.
Constitutive overexpression of CDK2 inhibits neuronal differentiation of rat pheochromocytoma PC12 cells.
J. Biol. Chem.
270:
23031-23037,
1995
11.
Dou, Q. P.,
A. H. Levin,
S. Zhao,
and
A. B. Pardee.
Cyclin E and cyclin A as candidates for the restriction point protein.
Cancer Res.
53:
1493-1497,
1993[Abstract].
12.
Durand, B.,
F. B. Gao,
and
M. Raff.
Accumulation of the cyclin-dependent kinase inhibitor p27/Kip1 and the timing of oligodendrocyte differentiation.
EMBO J.
16:
306-317,
1997
13.
Elledge, S. J.,
and
J. W. Harper.
Cdk inhibitors: on the threshold of checkpoints and development.
Curr. Opin. Cell Biol.
6:
847-852,
1994[Medline].
14.
Evers, B. M.,
T. C. Ko,
J. Li,
and
E. A. Thompson.
Cell cycle protein suppression and p21 induction in differentiating Caco-2 cells.
Am. J. Physiol.
271 (Gastrointest. Liver Physiol. 34):
G722-G727,
1996
15.
Gartel, A. L.,
M. S. Serfas,
M. Gartel,
E. Goufman,
G. S. Wu,
W. S. El-Deiry,
and
A. L. Tyner.
p21 (WAF1/CIP1) expression is induced in newly nondividing cells in diverse epithelia and during differentiation of the Caco-2 intestinal cell line.
Exp. Cell Res.
227:
171-181,
1996[Medline].
16.
Gordon, J. I.,
G. H. Schmidt,
and
K. A. Roth.
Studies of intestinal stem cells using normal, chimeric, and transgenic mice.
FASEB J.
6:
3039-3050,
1992
17.
Guan, K. L.,
C. W. Jenkins,
Y. Li,
C. L. O'Keefe,
S. Noh,
X. Wu,
M. Zariwala,
A. G. Matera,
and
Y. Xiong.
Isolation and characterization of p19INK4d, a p16-related inhibitor specific to CDK6 and CDK4.
Mol. Biol. Cell
7:
57-70,
1996[Abstract].
18.
Hannon, G. J.,
and
D. Beach.
p15INK4B is a potential effector of TGF-beta-induced cell cycle arrest.
Nature
371:
257-261,
1994[Medline].
19.
Hatakeyama, M.,
J. A. Brill,
and
G. R. Fink.
Collaboration of G1 cyclins in the functional inactivation of the retinoblastoma protein.
Genes Dev.
8:
1759-1771,
1994[Abstract].
20.
Hengst, L.,
V. Dulic,
J. M. Slingerland,
E. Lees,
and
S. I. Reed.
A cell cycle-regulated inhibitor of cyclin-dependent kinases.
Proc. Natl. Acad. Sci. USA
91:
5291-5295,
1994[Abstract].
21.
Hirai, H.,
M. F. Roussel,
J. Y. Kato,
R. A. Aashmun,
and
C. J. Sherr.
Novel INK4 proteins, p19 and p18, are specific inhibitors of the cyclin D-dependent kinases CDK4 and CDK6.
Mol. Cell. Biol.
15:
2672-2681,
1995[Abstract].
22.
Jones, B. A.,
and
G. J. Gores.
Physiology and pathophysiology of apoptosis in epithelial cells of the liver, pancreas, and intestine.
Am. J. Physiol.
273 (Gastrointest. Liver Physiol. 36):
G1174-G1188,
1997
23.
Kiyokawa, H.,
V. M. Richon,
R. A. Rifkind,
and
P. A. Marks.
Suppression of cyclin-dependent kinase 4 during induced differentiation of erythroleukemia cells.
Mol. Cell. Biol.
14:
7195-7203,
1994[Abstract].
24.
Ko, T. C.,
W. A. Bresnahan,
and
E. A. Thompson.
Intestinal cell cycle regulation.
Prog. Cell Cycle Res.
3:
43-52,
1997[Medline].
25.
Kranenburg, O.,
V. Scharnhorst,
A. J. van der Eb,
and
A. Zantema.
Inhibition of cyclin-dependent kinase activity triggers neuronal differentiation of mouse neuroblastoma cells.
J. Cell Biol.
131:
227-234,
1995[Abstract].
26.
Kranenburg, O.,
A. J. van der Eb,
and
A. Zantema.
Cyclin-dependent kinases and pRb: regulators of the proliferation-differentiation switch.
FEBS Lett.
367:
103-106,
1995[Medline].
27.
Markowitz, A. J.,
G. D. Wu,
A. Bader,
Z. Cui,
L. Chen,
and
P. G. Traber.
Regulation of lineage-specific transcription of the sucrase-isomaltase gene in transgenic mice and cell lines.
Am. J. Physiol.
269 (Gastrointest. Liver Physiol. 32):
G925-G939,
1995
28.
Messer, M.,
and
A. Dahlqvist.
A one step ultramicro method for the assay of intestinal disaccharidases.
Anal. Biochem.
14:
376-392,
1966[Medline].
29.
Morgan, D. O.
Principles of CDK regulation.
Nature
374:
131-134,
1995[Medline].
30.
Morse, L.,
D. Chen,
D. Franklin,
Y. Xiong,
and
S. Chen-Kiang.
Induction of cell cycle arrest and B cell terminal differentiation by CDK inhibitor p18(INK4c) and IL-6.
Immunity
6:
47-56,
1997[Medline].
31.
Nigg, E. A.
Cyclin-dependent protein kinases: key regulators of the eukaryotic cell cycle.
Bioessays
17:
471-480,
1995[Medline].
32.
Nurse, P.
Ordering S phase and M phase in the cell cycle.
Cell
79:
547-550,
1994[Medline].
33.
Pardee, A. B.
G1 events and regulation of cell proliferation.
Science
246:
603-608,
1989[Medline].
34.
Pinto, M.,
S. Robine-Léon,
M. D. Appay,
M. Kedinger,
N. Triadou,
E. Dussaulx,
B. Lacroix,
P. Simon-Assmann,
K. Haffen,
J. Fogh,
and
A. Zweibaum.
Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture.
Biol. Cell
47:
323-330,
1983.
35.
Podolsky, D. K.,
and
M. W. Babyatsky.
Growth and development of the gastrointestinal tract.
In: Textbook of Gastroenterology, edited by T. Yamada. Philadelphia, PA: Lippincott, 1995, p. 546-577.
36.
Ponder, B. A.,
G. H. Schmidt,
M. M. Wilkinson,
M. J. Wood,
M. Monk,
and
A. Reid.
Derivation of mouse intestinal crypts from single progenitor cells.
Nature
313:
689-691,
1985[Medline].
37.
Potten, C. S.
Epithelial cell growth and differentiation. II. Intestinal apoptosis.
Am. J. Physiol.
273 (Gastrointest. Liver Physiol. 36):
G253-G257,
1997
38.
Potten, C. S.,
and
M. Loeffler.
Stem cells: attributes, cycles, spirals, pitfalls and uncertainties. Lessons from the crypt.
Development
110:
1001-1020,
1990[Abstract].
39.
Serrano, M.,
G. J. Hannon,
and
D. Beach.
A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4.
Nature
366:
704-707,
1993[Medline].
40.
Sherr, C. J.
D-type cyclins.
Trends Biol. Sci.
20:
1887-1890,
1995.
41.
Sherr, C. J.,
and
J. M. Roberts.
Inhibitors of mammalian G1 cyclin-dependent kinases.
Genes Dev.
9:
1149-1163,
1995[Medline].
42.
Tikoo, R.,
P. Casaccia-Bonnefil,
M. V. Chao,
and
A. Koff.
Changes in cyclin-dependent kinase 2 and p27kip1 accompany glial cell differentiation of central glia-4 cells.
J. Biol. Chem.
272:
442-447,
1997
43.
Traber, P. G.
Differentiation of intestinal epithelial cells: lessons from the study of intestine-specific gene expression.
J. Lab. Clin. Med.
123:
467-477,
1994[Medline].
44.
Vachon, P. H.,
and
J.-F. Beaulieu.
Transient mosaic patterns of morphological and functional differentiation in the Caco-2 cell line.
Gastroenterology
103:
414-423,
1992[Medline].
45.
Vachon, P. H.,
N. Perreault,
P. Magny,
and
J.-F. Beaulieu.
Uncoordinated, transient mosaic patterns of intestinal hydrolase expression in differentiating human enterocytes.
J. Cell. Physiol.
166:
198-207,
1996[Medline].
46.
Van den Heuvel, S.,
and
E. Harlow.
Distinct roles for cyclin-dependent kinases in cell cycle control.
Science
262:
2050-2054,
1993[Medline].
47.
Weinberg, R. A.
The retinoblastoma protein and cell cycle control.
Cell
81:
323-330,
1995[Medline].
48.
Woo, M. S.,
I. Sanchez,
and
B. D. Dynlacht.
p130 and p107 use a conserved domain to inhibit cellular cyclin-dependent kinase activity.
Mol. Cell. Biol.
17:
3566-3579,
1997[Abstract].
49.
Yan, Y.,
J. Frisen,
M. H. Lee,
J. Massague,
and
M. Barbacid.
Ablation of the CDK inhibitor p57Kip2 results in increased apoptosis and delayed differentiation during mouse development.
Genes Dev.
11:
973-983,
1997[Abstract].
50.
Zweibaum, A.,
and
I. Chantret.
Human colon carcinoma cell lines as in vitro models for the study of intestinal cell differentiation.
In: Adaptation and Development of Gastrointestinal Function, edited by M. W. Smith,
and F. V. Sepulveda. Manchester, UK: Manchester University Press, 1989, p. 103-112.