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Intestinal Cell Differentiation: Cellular Mechanisms and the Search for the Perfect Model Focus on "Involvement of p21(WAF1/Cip1) and p27(Kip1) in intestinal epithelial cell differentiation"

B. Mark Evers

Department of Surgery, The University of Texas Medical Branch, Galveston, Texas 77555-0533


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THE MAMMALIAN INTESTINAL MUCOSA, a remarkable example of high efficiency and intricate complexity, is in a constant state of self-renewal, with complete mucosal turnover occurring every 3-5 days. Pluripotent stem cells, localized near the base of the crypt, are characterized by rapid proliferation and ascension along the crypt-villus axis, with cessation of proliferation and subsequent differentiation into one of the four primary cell types: absorptive enterocytes, goblet cells, Paneth cells, and enteroendocrine cells (1, 11). The cellular mechanisms regulating this highly regimented process of cell cycle arrest and differentiation have not been clearly defined. The elucidation of these mechanisms would be useful not only in delineating normal cell processes leading to the differentiated phenotype but also in providing information regarding abnormal processes, such as neoplasia formation, that can occur when these mechanisms go awry. Although an area of active research interest, this field of investigation has been hampered by the lack of cellular models that accurately reflect what is occurring in vivo.

For the most part, investigators have relied on human colon cancer cell lines that possess the property of differentiating to an enterocyte-like phenotype to investigate mechanisms of normal cellular differentiation (16). For example, two well-characterized cell lines include the Caco-2 and HT-29 colon cancer cell lines that differentiate either spontaneously or in response to changes in media or differentiating agents, such as sodium butyrate. In the current article in focus by Tian and Quaroni (Ref. 14, see p. C1245 in this issue), a novel approach has been utilized to generate a human fetal intestinal cell line (tsFHI), immortalized with a temperature-sensitive SV40 T-antigen (SV40 T-Ag) (12). At the permissive temperature (i.e., 32°C), tsFHI cells actively proliferate and display crypt cell markers. However, with a shift to the nonpermissive temperature (i.e., 39°C), the cells undergo irreversible growth arrest and shift to a differentiated phenotype, as noted by expression of differentiated brush-border membrane proteins, including aminopeptidase N, dipeptidyl-peptidase IV, and sucrase-isomaltase. Whereas other temperature-sensitive cell lines have been derived from the rat and mouse (10, 15), the tsFHI cell line was derived from human intestinal cells and obviously is quite attractive in this regard. This cell line recapitulates, in a number of respects, the well-coordinated in vivo process of active proliferation with subsequent growth arrest and a shift to a differentiated phenotype. Although this cellular model is appealing in its approach, the results obtained must be interpreted with some caution, since the functional SV40 T-Ag may interfere with certain cellular signaling pathways. That being said, this novel cell line should still prove to be quite beneficial and valuable in further delineating the pathways that trigger intestinal cells to differentiate.

Cellular factors that determine whether cells continue to proliferate or cease dividing and differentiate appear to function during the first gap phase (G1) of the cell cycle. Progression through the cell cycle is regulated by a highly conserved family of serine/threonine protein kinases that are composed of a regulatory subunit (the cyclins) and a catalytic subunit [the cyclin-dependent kinases (Cdks)] (9, 13). Progression through the G1 phase requires association of D-type cyclins with Cdk4 and Cdk6, whereas cyclin E binding to Cdk2 is required for the G1/S transition. The activities of the Cdks can be inhibited by the binding of Cdk-inhibitory proteins, of which two families have been identified (4). The first family consists of universal inhibitors of the cyclin/Cdk complexes and includes p21(WAF1/Cip1) and p27(Kip1/Pic2). Another family appears to selectively bind and inhibit Cdk4 and Cdk6 and includes p15(Ink4b), p16(Ink4a), p18(Ink4c), and p19(Ink4d). In addition, important targets of Cdk4 and Cdk2 include the retinoblastoma protein (pRb) and the pRb-related proteins, p107 and p130 (7). Previous studies have shown that differentiation of the Caco-2 cell line is associated with a suppression of Cdk2 and Cdk4 activities, which precede the G1/S cell cycle block and most likely contribute to this process (3). In addition, induction of the Cdk inhibitor protein p21(WAF1/Cip1) contributes to this initial cell cycle block (3, 5, 8). The maintenance of the differentiated phenotype appears to then involve downregulation of cyclin and Cdk protein expression and inhibition of cyclin/Cdk complex formation. Findings in this study by Tian and Quaroni (14) using the tsFHI cell line corroborate many of the findings noted with Caco-2 cell differentiation and, moreover, greatly expand our current knowledge regarding the complicated interactions that occur with intestinal cell differentiation. The authors further emphasize that the increase of p21(WAF1/Cip1) is rapid and transient and may be involved in the early stages of differentiation; however, involvement of p21(WAF1/Cip1) in the maintenance of the differentiated phenotype is probably not required. These results are consistent with findings in the differentiated Caco-2 cell line as well as in transgenic mice that lack the p21 gene (2). A working hypothesis is proposed regarding the involvement of these cell cycle-related proteins in intestinal cell differentiation. Thus multiple (and potentially redundant) mechanisms are likely responsible for the initial cell cycle arrest and the maintenance of differentiated phenotype.

In summary, the report by Tian and Quaroni (14) is important for the assessment of intestinal cell differentiation from several different standpoints. First, it represents the use of a novel human intestinal cell line that should be useful in further delineating signaling pathways that are important for the switch from active proliferation to differentiation. While not perfect, the results using this cell line will be highly illuminating. In addition, the authors further emphasize that multiple and potentially redundant mechanisms are at work to achieve and maintain a differentiated phenotype. Correlation of findings using this cell line with transgenic models of intestinal differentiation (6) should prove immensely useful in unraveling the complex series of events that lead to the remarkably efficient and orderly process of intestinal cell proliferation and differentiation.


    FOOTNOTES

Address for reprint requests and other correspondence: B. M. Evers, Department of Surgery, The University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0533 (E-mail: mevers{at}utmb.edu).


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1.   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].

2.   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].

3.   Ding, Q.-M., T. C. Ko, and B. M. Evers. Caco-2 intestinal cell differentiation is associated with G1 arrest and suppression of CDK2 and CDK4. Am. J. Physiol. 275 (Cell Physiol. 44): C1193-C1200, 1998[Abstract/Free Full Text].

4.   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].

5.   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[Abstract/Free Full Text].

6.   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[Abstract/Free Full Text].

7.   Kranenburg, O., A. J. van der Eb, and Z. Zantema. Cyclin-dependent kinases and pRb: regulators of the proliferation-differentiation switch. FEBS Lett. 367: 103-106, 1995[Medline].

8.   Litvak, D. A., B. M. Evers, K. O. Hwang, M. R. Hellmich, T. C. Ko, and C. M. Townsend, Jr. Butyrate-induced differentiation of Caco-2 cells is associated with apoptosis and early induction of p21Waf1/Cip1 and p27Kip1. Surgery 124: 161-170, 1998[Medline].

9.   Morgan, D. O. Principles of CDK regulation. Nature 374: 131-134, 1995[Medline].

10.   Paul, E. C. A., J. Hochman, and A. Quaroni. Conditionally immortalized intestinal epithelial cells: novel approach for study of differentiated enterocytes. Am. J. Physiol. 265 (Cell Physiol. 34): C266-C278, 1993[Abstract/Free Full Text].

11.   Potten, C. S., and M. Loeffler. Stem cells: attributes, cycles, spirals, pitfalls and uncertainties. Lessons from the crypt. Development 110: 1001-1020, 1990[Abstract].

12.   Quaroni, A., and J.-F. Beaulieu. Cell dynamics and differentiation of conditionally immortalized human intestinal epithelial cells. Gastroenterology 113: 1198-1213, 1997[Medline].

13.   Sherr, C. J. D-type cyclins. Trends Biochem. Sci. 20: 1887-1890, 1995.

14.   Tian, J. Q., and A. Quaroni. Involvement of p21(WAF1/Cip1) and p27(Kip1) in intestinal epithelial cell differentiation. Am. J. Physiol. 276 (Cell Physiol. 45): C1245-C1258, 1999[Abstract/Free Full Text].

15.   Whitehead, R. H., P. E. VanEeden, M. D. Noble, P. Ataliotis, and P. S. Jat. Establishment of conditionally immortalized epithelial cell line from both colon and small intestine of adult H-2Kb-tsA58 transgenic mice. Proc. Natl. Acad. Sci. USA 90: 587-591, 1993[Abstract].

16.   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 Univ. Press, 1989, p. 103-112.


Am J Physiol Cell Physiol 276(6):C1243-C1244
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society




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