1. Volume 129, Issue 6, Pages 1875-1888 (December 2005)
Clostridium difficile Toxin A–Induced Colonocyte Apoptosis Involves p53-Dependent p21(WAF1/CIP1) Induction via p38 Mitogen-Activated Protein Kinase 
Ho Kim, Efi Kokkotou, Xi Na, Sang Hoon Rhee, Mary P. Moyer, Charalabos Pothoulakis, J. Thomas Lamont 
Gastroenterology 
Volume 129, Issue 6, Pages (December 2005)
DOI: /j.gastro
Copyright © 2005 American Gastroenterological Association Terms and Conditions

2. Figure 1
C difficile toxin A induces apoptosis and cell cycle arrest in human colonocytes. (A) Colonocytes were incubated with toxin A (3 nmol/L) for the indicated times. Identical fields were observed for TUNEL-positive (green) and PI (red) counterstain for all nuclei. (B) Colonocytes were incubated with 3, 30 nmol/L toxin A for 48 hours. DNA laddering was run on 1% agarose gels along with marker. (C) Quantitation of cell viability by MTT assay. (D) Colonocytes were incubated with toxin A for the indicated time points, and whole cell lysates were prepared. Cytosolic extracts were also prepared to measure cytosolic cytochrome c. Cell lysates were resolved on 15% polyacrylamide gels. (E) Colonocytes were incubated with toxin A or staurosporine (1 μmol/L) for the indicated times. (F) Subconfluent cultures of colonocytes were incubated with toxin A for 12 or 48 hours. Cell cycle phases were determined by flow cytometry of cellular DNA content. Results are representative of 3 independent experiments.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

3. Figure 2
Toxin A increases expression of p21(WAF1/CIP1) and activation of p53 and p38. (A, C, and D) Colonocytes were incubated with 3 nmol/L toxin A for the indicated times. Cell lysates resolved on 10% polyacrylamide gels and probed with the indicated antibodies. (B) Total RNA was isolated and expression of p21(WAF1/CIP1) and β-actin messenger RNA were determined by Northern blotting. Results are representative of 3 independent experiments. (E) Colonocytes were exposed to toxin A, and TNF-α levels in the conditioned medium were measured by enzyme-linked immunosorbent assay. The bars represent the mean ± SD of 3 independent experiments.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

4. Figure 3
Toxin A–induced phosphorylation of p53/p38, induction of p21(WAF1/CIP1)/Bak, and colonocyte apoptosis are independent of Rho monoglucosylation by toxin A. (A) Time course and concentration dependence of UDP-dialdehyde treatment on toxin A–induced Rho ribosylation. C botulinum C3-catalyzed adenosine diphosphate ribosylation of Rho was assayed. (B and C) Colonocytes were incubated for 24 hours with medium (control), UDP-dialdehyde (1 mmol/L), toxin A (3 nmol/L), or toxin A together with UDP-dialdehyde, and then DNA fragmentation was also measured by PI staining and FACS analysis. (C) *P < .001 vs nonstimulated cells; **P < .005 vs toxin A–stimulated cells. (D) Colonocytes were pretreated with UDP-dialdehyde for 1 hour before stimulation with toxin A for 3 and 36 hours. (E) Alkylated toxin A has no glucosyltransferase activity. Colonocytes were incubated with alkylated toxin A (3 nmol/L) or with either native toxin A (3 nmol/L) or alkylated toxin A (15 nmol/L) plus native toxin A for 60 minutes. (F) Colonocytes were incubated with 15 nmol/L alkylated toxin A, with either 3 nmol/L native toxin A (TxA) or with alkylated toxin A plus native toxin A for 24 hours. (G) Colonocytes were incubated with alkylated toxin A and native toxin A for 24 hours. *P < .001 vs nonstimulated cells. (H) DNA-laddering experiments with alkylated toxin A. (I) Colonocytes were incubated with alkylated toxin A for the indicated times.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

5. Figure 4
p38-dependent p21(WAF1/CIP1) induction in colonocytes exposed to toxin A. (A) Colonocytes were treated with either medium (control) or toxin A alone or toxin A together with either dimethyl sulfoxide, SB (10 μmol/L), AG490 (50 μmol/L), or SP (10 μmol/L). (B) Total RNA was isolated from colonocytes treated as in A, and expression of p21(WAF1/CIP1) and β-actin messenger RNA were determined by Northern blotting. (C) Colonocytes were transiently transfected with the p21(WAF1/CIP1) promoter and a p38 dominant negative mutant (p38 DN) at the indicated concentrations for 24 hours. Results are expressed as mean ± SEM of 3 experiments per group. *P < .001 vs nonstimulated cells; **P < .005 vs toxin A–stimulated cells. (D) Colonocytes were incubated with toxin A and/or control goat immunoglobulin G, TGF-α, and TGF-β neutralizing antibodies for 6 hours.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

6. Figure 5
Toxin A activates p38-phosphorylated p53 at serine 46 but not at serine 15. (A) Active p38 was prepared using antibody against phospho-p38 from colonocytes exposed to toxin A for 1 hour. p38-containing immune complexes were incubated with [32P]ATP and full-length GST-p53 protein (5 μg), and samples were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography. (B) To define the phosphorylation site of p53, kinase assay was performed in the presence of recombinant GST-p53 protein (5 μg), 200 mmol/L ATP, and p38-containing immune complex. The phosphorylated proteins were detected with antibodies directed against p53, and phospho-p53 at serine 15 or serine 46. (C) Kinase assay was performed using recombinant active p38 in the presence of GST-p53 protein, GST-c-Jun (1-79) protein, and ATP. The phosphorylated proteins were detected with antibodies against p53 and phospho-p53 at serine 46. (D) Colonocytes were treated with either medium, or toxin A alone, or toxin A together with either the chemical inhibitors indicated above.
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
p38-dependent activation of p53 regulates the transcription of p21(WAF1/CIP1) in colonocytes exposed to toxin A. (A) Colonocytes were transfected with full-length and various deletion mutants of p21(WAF1/CIP1) promoter constructs. Cells were further incubated with toxin A for 24 hours, and cell extracts were prepared to measure p21(WAF1/CIP1) promoter activity. Results are expressed as mean ± SEM of 3 experiments per group. *P < .005 vs nonstimulated cells. (B) Colonocytes were incubated with toxin A for the indicated times. Nuclear extracts (2 μg) were incubated with 1 ng of radiolabeled probe at room temperature for 30 minutes. “Probe” indicates that no nuclear extracts were added. (C) Nuclear extracts from cells treated with toxin A for 1 hour were incubated with radiolabeled probe for 30 minutes and rabbit control immunoglobulin G; p53 antibodies were added to the reaction mixture before the addition of labeled probe (lanes 3–4). (D) Toxin A–induced DNA binding affinity of p53 is regulated by p38 MAPK.
Gastroenterology  , DOI: ( /j.gastro )
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8. Figure 7
Lack of toxin A induction of p21(WAF1/CIP1) and activation of apoptotic molecules in p53-silenced colonocytes. (A) Colonocytes were stably transfected with the pSuppressorNeo-p53 plasmid expressing p53-RNAi target sequences. (B) Negative control or p53 knockdown cells were incubated with toxin A for 3 hours and the expression of p21(WAF1/CIP1), p27, and p38 was determined. (C) The p21(WAF1/CIP1) promoter construct (−2.4 kilobases) was transfected into negative control cells or the p53-silenced cell lines. #P < .005 vs nonstimulated cells; *P < .001 vs toxin A–stimulated control cells. (D) Subconfluent cultures of negative control and p53-silenced cells were incubated with toxin A for 24 hours. Cell cycle phases were determined by flow cytometry. (E) Negative control and p53 knockdown cells were incubated with toxin A for 36 hours. (F) Negative control and p53 knockdown cells were incubated with toxin A for 24 hours. DNA fragmentation was measured by PI staining and FACS analysis. *P < .005 vs toxin A–stimulated control cells. (G) Negative control cells and the p53 knockdown cells were treated with toxin A, and cell viability was measured by MTT assay.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

9. Figure 8
Overexpression of p21(WAF1/CIP1) is associated with increased toxin A–induced colonocyte apoptosis. Colonocytes were transiently transfected with pcDNA3 and pcDNA3-p21(WAF1/CIP1) and processed as explained below. (A) Cell extracts were prepared to measure exogenous p21(WAF1/CIP1) expression. Cell cycle distribution of mock transfected cells (left) or p21(WAF1/CIP1) transfected cells (right) was determined by flow cytometry. (B) Forty-eight hours after transfection of colonocytes with p21(WAF1/CIP1), cells were incubated with toxin A for the indicated time intervals. (C and D) Cells transfected with pcDNA3 or pcDNA3-p21(WAF1/CIP1) were exposed to toxin A for indicated times, and DNA fragmentation and cell viability were measured by PI staining and FACS analysis or MTT assay.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

10. Figure 9
Pharmacologic inhibition of p38 MAPK reduces toxin A–induced p21(WAF1/CIP1) activation, apoptosis, and villi destruction in mouse ileum. (A) Blockage of p38 by SB prevents toxin A–induced apoptosis of ileal mucosa. Apoptotic cells (green) were evaluated by TUNEL staining and counterstained with PI (red). (B) Tissues were exposed to antibodies against p21(WAF1/CIP1) and then to a fluorescein isothiocyanate–conjugated anti-rabbit immunoglobulin G. Frozen tissue sections were exposed to PI to stain cellular nuclei (original magnification 200×). (C) Sections were double stained with anti-p21(WAF1/CIP1) (green) and anti-cytokeratin (red) antibodies. Colocalized expression is indicated by the yellow color in the merge panel. (D) Tissue lysates were resolved on 15% polyacrylamide gels and probed with a p21(WAF1/CIP1) and β-actin antibody (n = 3 mice per group). (E) SB substantially blocked toxin A–induced epithelial cell destruction. Frozen tissue slides were stained with PI to stain cellular nuclei (original magnification 100×).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions