Response of small intestinal epithelial cells to acute disruption of cell division through CDC25 deletion - PubMed (original) (raw)

Response of small intestinal epithelial cells to acute disruption of cell division through CDC25 deletion

Gwanghee Lee et al. Proc Natl Acad Sci U S A. 2009.

Abstract

The CDC25 protein phosphatases (CDC25A, B, and C) drive cell cycle transitions by activating key components of the cell cycle engine. CDC25A and CDC25B are frequently overproduced in human cancers. Disruption of Cdc25B or Cdc25C individually or in combination has no effect on mouse viability. Here we report that CDC25A is the only family member to provide an essential function during early embryonic development, and that other family members compensate for its loss in adult mice. In contrast, conditional disruption of the entire family is lethal in adults due to a loss of small intestinal epithelial cell proliferation in crypts of Lieberkühn. Cdc25 loss induced Wnt signaling, and overall crypt structures were preserved. In the face of continuous Wnt signaling, nearly all crypt epithelial progenitors differentiated into multiple cell lineages, including crypt base columnar cells, a proposed stem cell. A small population of Musashi/Dcamkl-1/nuclear beta-catenin-positive epithelial cells was retained in these crypts. These findings have implications for the development of novel, less cytotoxic cancer chemotherapeutic drugs that specifically target the cell cycle.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Loss of homeostasis in small intestines of TKO mice. All graphical data are presented as mean ± SEM. An asterisk indicates a significant difference from WT mice injected with OHT as determined by the 2-tailed Student _t_- test. The α level is .05. Individual P values are presented in each panel. (A) Significant shortening of the small intestine in TKO mice. From the top, P values are .008, .003, and .004. Intestinal lengths did not vary significantly between the AKO mice and the WT mice (P = .07). (B) Significant shortening of villi in the small intestine of TKO mice. From the top, P values are .0004, .008, and .0001. (C) Shortened villi in the small intestine of TKO mice. (Scale bar: 0.5 mm.) The insets show higher magnifications of boxed areas. (D) Lack of phenotype in the small intestine of mice disrupted for Cdc25A. The mice were killed 90 days after the final OHT injection. (E) Absence of mitotic figures in crypt cells of TKO mice. (Scale bar: 20 μm.) Crypt borders are depicted with blue hatched lines. (F) Absence of BrdU-positive cells in crypts of TKO mice. (Scale bar: 20 μm.) Crypt borders are depicted with black hatched lines. (G) Crypts within the proximal portion of the small intestine of OHT mice were examined for epithelial cells, Paneth cells, mitotic cells (presence of mitotic figures), and apoptotic cells (presence of fragmented nuclei). Areas containing Brunner's gland were excluded from analysis. Twenty crypts were counted per mouse, and 2 mice of each genotype were evaluated. Similar patterns were observed in mid and distal portions of the small intestine (data not shown).

Fig. 2.

Fig. 2.

G1 and G2 cell cycle arrest of epithelial cells in the small intestine of TKO mice. (A) Loss of CDC25 causes intestinal epithelial cells to arrest in the G1 and G2 phases of the cell cycle. R26CreER_T;A f/−_BCKO mice were injected with OHT for 1, 2, or 3 days and killed 24 h after the last injection to generate TKO mice. One hour before sacrifice, the mice were injected with BrdU to label S-phase cells. Green, geminin; red, BrdU; blue, DAPI. Insets display individual crypts at higher magnifications. Crypt borders are depicted with white hatched lines. The arrow in the WT image denotes a G2 cell in the crypt. (Scale bar: 10 μm.) (B) G1-, S-, and G2-phase cells were quantitated from the images shown in (D). Total nuclei counted: WT, 502; TKO, 416 on day 2, 399 on day 3, and 409 on day 4.

Fig. 3.

Fig. 3.

Morphology and composition of intestinal crypts in TKO mice. (A–D) Intestinal sections were stained with PAS/Alcian blue to label Paneth cells and goblet cells. Sections from WT (A) and TKO (B–D) mice are shown. The inset in (B) is magnified in (C). In (A), asterisks indicate CBC cells. In (D), asterisks indicate immature Paneth cells. Crypt borders are indicated with black hatched line. (Scale bar: 10 μm.) (E and F) Intestinal sections were stained with the enterocyte-specific marker L-Fabp. Sections from WT (E) and TKO (F) mice are shown. Green, L-Fabp; blue, nuclei (DAPI). Hatched lines denote crypt margins. (Scale bar: 100 μm.) (G) Musashi-1– and Dcamkl-1–positive cells are maintained in TKO mice. Green (Upper), Musashi-1; green (Lower), Dcamkl-1; red, BrdU; blue, DAPI. (Scale bars: 20 μm.) Crypt borders are outlined. Arrows indicate cells staining negative for BrdU but positive for Musashi-1 or Dcamkl-1. (H) The number and location of 28 Mushashi-1–positive cells were determined in TKO mice. The cell at the base of the crypt was defined as position 1.

Fig. 4.

Fig. 4.

Enhanced Wnt signaling and differentiation in crypts of TKO mice. (A and B) Tissue sections of small intestines prepared from WT (A) and TKO (B) mice were stained with antibodies specific for β-catenin (green) and lysozyme (red). Nuclei were stained with DAPI (blue). Arrows indicate cells staining positive for nuclear β-catenin. (Scale bar: 10 μm.) (C–G) Small intestines were dissected and processed for transmission electron microscopy. (D) An EM section of crypt from a WT mouse and higher magnification (C) showing a CBC cell between 2 mature Paneth cells, marked with an asterisk in (D). (E) EM sections of crypt from a TKO mouse. The inset in (E) is shown at higher magnification in (F). The arrow indicates an immature goblet cell with characteristic apical mucinogenic granules. In (F), a cell with stem-like morphology above the Paneth cell compartment is marked with an asterisk. (G) CBC cells differentiated along the Paneth cell lineage as marked by the appearance of small, electron-dense secretory granules. In (G), asterisks indicate the position of the intestinal lumen. [Scale bar: 10 μm (D and E); 2 μm (F and G).]

References

    1. Lee MS, et al. Cdc25+ encodes a protein phosphatase that dephosphorylates p34cdc2. Mol Biol Cell. 1992;3:73–84. - PMC - PubMed
    1. Dunphy WG, Kumagai A. The CDC25 protein contains an intrinsic phosphatase activity. Cell. 1991;67:189–196. - PubMed
    1. Gautier J, Solomon MJ, Booher RN, Bazan JF, Kirschner MW. CDC25 is a specific tyrosine phosphatase that directly activates p34cdc2. Cell. 1991;67:197–211. - PubMed
    1. Sebastian B, Kakizuka A, Hunter T. Cdc25M2 activation of cyclin-dependent kinases by dephosphorylation of threonine-14 and tyrosine-15. Proc Natl Acad Sci U S A. 1993;90:3521–3524. - PMC - PubMed
    1. Strausfeld U, et al. Dephosphorylation and activation of a p34cdc2/cyclin B complex in vitro by human CDC25 protein. Nature. 1991;351:242–245. - PubMed

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