A vitamin D3 analog induces a G1-phase arrest in CaCo-2 cells by inhibiting cdk2 and cdk6: roles of cyclin E, p21Waf1, and p27Kip1.
Previous studies by our laboratory have shown that a noncalcemic fluorinated analog of 1alpha,25-dihydroxyvitamin D3, 1alpha,25-dihydroxy-16-ene-23-yne-26,27-hexafluorocholcal ciferol (F6-D3), significantly reduced the frequency of colonic adenomas and completely abolished the development of colonic adenocarcinomas in rats treated with azoxymethane. The mechanisms involved in this analog's chemopreventive actions, however, remain unclear. In the present study, we now show that although both 1alpha,25-dihydroxyvitamin D3 and F6-D3 inhibited the proliferation of CaCo-2 cells, a human colonic adenocarcinoma cell line, by increasing their doubling times, only F6-D3 caused an arrest of these cells in the G1 phase of their cell cycle. This arrest was accompanied by an increase in the expression of the cyclin-dependent kinase (cdk) inhibitor proteins, p2Waf1 and p27Kip1, which served to decrease the activity of cyclin-dependent kinase 2 and cyclin-dependent kinase 6, whereas the expression and phosphorylation of pRB were unchanged. In contrast to the increased expression of these cdk inhibitors, the expression of cyclin E was decreased, which further inhibited the activity of cyclin-dependent kinase 2. Collectively, the inhibition of these cyclin-dependent kinases served to arrest the CaCo-2 cells, independent of changes in pRB. Furthermore, antibody neutralization studies suggest that transforming growth factor-beta may mediate the coassociations between cdk2 and p27Kip1 and cyclin E induced by F6-D3. These data indicate that cell cycle arrest may, at least in part, underlie the chemopreventive actions of F6-D3 observed in the azoxymethane model of colon cancer. Furthermore, if the antiproliferative action observed in CaCo-2 cells also occurs in human colonic epithelium, F6-D3 may have chemopreventive potential against human colon cancer, as well.
Scaglione-Sewell
B A
BA
Department of Medicine, The University of Chicago, Illinois 60637, USA.
Bissonnette
M
M
Skarosi
S
S
Abraham
C
C
Brasitus
T A
TA
D016588:Anticarcinogenic Agents / Q000494:pharmacology; D042846:CDC2-CDC28 Kinases; D018938:Caco-2 Cells / Q000473:pathology*; D002117:Calcitriol / Q000031:analogs & derivatives* / Q000494:pharmacology*; D018797:Cell Cycle Proteins; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D019927:Cyclin E / Q000502:physiology; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016213:Cyclins / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016193:G1 Phase / Q000187:drug effects*; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D025521:Tumor Suppressor Proteins; D005466:Fluorocarbons
D016588:Anticarcinogenic Agents; D042846:CDC2-CDC28 Kinases; D002117:Calcitriol; D018797:Cell Cycle Proteins; D019927:Cyclin E; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases; D016213:Cyclins; D004791:Enzyme Inhibitors; D008869:Microtubule-Associated Proteins; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D025521:Tumor Suppressor Proteins; C493956:CDKN1A protein, human; C495894:CDK2 protein, human; C495900:CDK4 protein, human; C495911:CDK6 protein, human; C040488:26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3; D005466:Fluorocarbons