Downregulation of cyclin D1 alters cdk 4- and cdk 2-specific phosphorylation of retinoblastoma protein.
Progression of cells through the G1 phase of the cell cycle requires the assembly and activation of specific cyclin:cyclin-dependent kinase (cdk) complexes in a tightly regulated, sequential fashion. To more clearly define the temporal events leading to the G1/S transition, sequential changes in the expression of cyclin E and cdks 2, 4, and 6, as well as the phosphorylation of the retinoblastoma protein (pRb), were assayed in RA28 cells, a variant of human colon cancer RKO cells which were modified by transfection of an ecdysone-inducible antisense (AS) CD1 expression system. Induction of cyclin D1 antisense mRNA by the ecdysteroid, ponasterone A, resulted in a 55% decrease in cyclin D1 mRNA and a 58% decrease in CD1 protein levels. There was a 2.4-fold decrease in the ratio of hyperphosphorylated pRb (ppRb) to hypophosphorylated pRb, as well as a 60-75% decrease in cdk 2- and cdk 4-specific phosphorylated pRb proteins. Of interest, cyclin E-dependent phosphorylation (cdk2) decreased 2.5-fold at 3 h despite only a 30% decrease in cyclin E protein level. Levels of cdk 2, cdk 4, and cdk 6 decreased 40-70%, while levels of cyclin A and B were unaffected by induction of CD1 antisense. Induction of a CD1 antisense gene in a human colon cancer cell line resulted in rapid, concomitant changes in CD1 mRNA and protein, cyclin E, cdk2, cdk4, and cdk6, as well as the ratio of ppRb to pRb. In this system, growth regulatory events are tightly regulated and the perturbed expression of a single protein, CD1, rapidly alters expression of multiple regulatory proteins involved in the G1/S transition phase of cell cycle progression.
Yu
B
B
Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, 10461, USA.
Lane
M E
ME
Pestell
R G
RG
Albanese
C
C
Wadler
S
S
D015153:Blotting, Western; D042846:CDC2-CDC28 Kinases; D003110:Colonic Neoplasms / Q000378:metabolism; D019938:Cyclin D1 / Q000235:genetics / Q000378:metabolism*; D019927:Cyclin E / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D015536:Down-Regulation / Q000187:drug effects; D004441:Ecdysterone / Q000031:analogs & derivatives* / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D016372:RNA, Antisense / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011987:Receptors, Steroid / Q000235:genetics; D016160:Retinoblastoma Protein / Q000378:metabolism*; D013379:Substrate Specificity; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection; D014407:Tumor Cells, Cultured
D019927:Cyclin E; D011518:Proto-Oncogene Proteins; D016372:RNA, Antisense; D012333:RNA, Messenger; D011987:Receptors, Steroid; D016160:Retinoblastoma Protein; C055469:ecdysone receptor; D019938:Cyclin D1; D004441:Ecdysterone; C008999:ponasterone A; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495900:CDK4 protein, human; C495911:CDK6 protein, human; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6; D018844:Cyclin-Dependent Kinases