Butyrate-induced differentiation of Caco-2 cells is associated with apoptosis and early induction of p21Waf1/Cip1 and p27Kip1.
BACKGROUND
Intestinal mucosal turnover is a process of proliferation, differentiation, and apoptosis; the mechanisms remain largely undefined. The purpose of our study was to (1) assess the relationship between apoptosis and enterocyte differentiation and (2) determine whether the cell-cycle inhibitors, p21Waf1/Cip1 and p27Kip1, or the apoptosis inhibitors, Bcl-2 and Bcl-XL, may be involved.


METHODS
Gut-derived Caco-2 cells were treated with sodium butyrate. Apoptosis was assessed by Hoechst stain, DNA laddering, and annexin V assay; differentiation was determined by alkaline phosphatase and sucrase activity. RNA and protein were analyzed for expression of p21Waf1/Cip1, p27Kip1, and members of the Bcl-2 family.


RESULTS
Treatment of Caco-2 cells with sodium butyrate resulted in the concomitant induction of both differentiation (increased alkaline phosphatase and sucrase activity) and apoptosis. Increased levels of p21Waf1/Cip1 and p27Kip1 mRNA and protein were detected at 24 hours, occurring before apoptosis or differentiation; decreased mRNA levels of Bcl-2 and Bcl-XL were noted at 24 hours.


CONCLUSIONS
Differentiation and apoptosis occurred simultaneously in Caco-2 cells, suggesting that apoptosis may be linked to enterocyte differentiation. The induction of p21Waf1/Cip1 and p27Kip1 and the down-regulation of Bcl-2 and Bcl-XL further suggest a link between the cell-cycle mechanisms regulating enterocyte differentiation and apoptosis.
Department of Surgery, University of Texas Medical Branch, Galveston 77555-0533, USA.
D A
DA
Litvak
B M
BM
Evers
K O
KO
Hwang
M R
MR
Hellmich
T C
TC
Ko
C M
CM
Townsend
D000469:Alkaline Phosphatase / Q000032:analysis / Q000378:metabolism; D017304:Annexin A5 / Q000032:analysis; D017209:Apoptosis / Q000187:drug effects*; D002087:Butyrates / Q000494:pharmacology*; D020148:Butyric Acid; D018938:Caco-2 Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D018797:Cell Cycle Proteins; D002454:Cell Differentiation / Q000187:drug effects; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D016213:Cyclins / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000378:metabolism*; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D016147:Genes, Tumor Suppressor / Q000502:physiology; D006633:Histamine Antagonists / Q000494:pharmacology*; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000235:genetics*; D008871:Microvilli / Q000201:enzymology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D012260:Ribonucleases; D013393:Sucrase / Q000032:analysis / Q000378:metabolism; D025521:Tumor Suppressor Proteins
D017304:Annexin A5; D002087:Butyrates; C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004791:Enzyme Inhibitors; D006633:Histamine Antagonists; D008869:Microtubule-Associated Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D025521:Tumor Suppressor Proteins; D020148:Butyric Acid; D050760:Cyclin-Dependent Kinase Inhibitor p27; D012260:Ribonucleases; D000469:Alkaline Phosphatase; D013393:Sucrase