Modulation of CDK2-AP1 (p12(DOC-1)) expression in human colorectal cancer.
We have previously demonstrated an association between microsatellite instability and decreased CDK2-AP1 (p12(DOC-1)) expression in human colorectal cancer (CRC) cell lines. In those same studies, induction of CDK2-AP1 expression promoted both cell cycle arrest and apoptosis. The goals of our present study were to better understand the mechanisms leading to reduced CDK2-AP1 expression in microsatellite unstable (MSI) CRC and to study further the effect of CDK2-AP1 modulation on cell proliferation and apoptosis utilizing RNA interference (RNAi) techniques. We used direct sequencing to screen for mutations of the poly (T)8 microsatellite-like region in the 3' end of the CDK2-AP1 gene in 24 CRC cell lines. We then utilized an in vitro human mismatch repair (MMR) recombinant system to assess for correction of the mutation and changes in CDK2-AP1 expression secondary to hMLH1 transfection. We also investigated the effect of CDK2-AP1 modulation in four settings: (1) native CDK2-AP1 absence, (2) endogenous CDK2-AP1 expression, (3) RNAi-induced CDK2-AP1 inhibition and (4) induced CDK2-AP1 over expression. The mutation - del T poly (T)8 - at the 3' end of the CDK2-AP1 gene was found in 3/12 (25%) of MSI CRC cell lines, but in none of the microsatellite stable samples (0/12). Interestingly, when wild-type MMR protein - MLH1 - was induced in an in vitro human recombinant system, the del T poly (T)8 mutation was reversed and CDK2-AP1 expression increased. RNAi-mediated CDK2-AP1 inhibition was associated with decreased apoptosis and increased cell proliferation in CDK2-AP1-non deficient CRC cell lines. We conclude that mutations in the microsatellite-like sequence of the CDK2-AP1 gene in MSI CRC are associated with decreased CDK2-AP1 expression. In addition, modulation of CDK2-AP1 expression in human CRC alters cell proliferation and apoptosis.
Yuan
Ziquan
Z
Department of Molecular Genetics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Ullmann 707, New York, NY 10461, USA.
Gaba
Anu G
AG
Kent
Tara Sotsky
TS
Bennett
Anna
A
Miller
Agnes
A
Weber
Thomas K
TK
D017209:Apoptosis / Q000502:physiology; D020137:Base Pair Mismatch; D001483:Base Sequence; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D015179:Colorectal Neoplasms / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D018895:Microsatellite Repeats / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D034622:RNA Interference; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D025521:Tumor Suppressor Proteins / Q000096:biosynthesis* / Q000235:genetics*
C096151:CDK2AP1 protein, human; D012333:RNA, Messenger; D025521:Tumor Suppressor Proteins