The APC tumor suppressor inhibits DNA replication by directly binding to DNA via its carboxyl terminus.
OBJECTIVE
The APC tumor suppressor is well known for its ability to regulate Wnt signaling through mediation of beta-catenin levels in the cell. Transient over expression of the tumor suppressor gene APC in colon cancer cells prevents entry into S phase of the cell cycle, a phenotype only partially restored by cotransfection of a transcriptionally active form of beta-catenin. In an attempt to define its transcription-independent tumor suppressor functions, we tested whether APC directly affects DNA replication.


METHODS
A transcriptionally quiescent in vitro DNA replication system, the polymerase chain reaction, DNA binding assays, and transient transfections in colon cancer cell lines were used to determine the effects of APC on DNA replication and the mechanism by which it works.


RESULTS
We report that exogenous full-length APC inhibits replication of template DNA through a function that maps to amino acids 2140-2421, a region of the protein commonly lost by somatic or germline mutation. This segment of APC directly interacts with DNA, while mutation of the DNA-binding S(T)PXX motifs within it abolishes DNA binding and reduces inhibition of DNA replication. Phosphorylation of this segment by cyclin-dependent kinases also reduces inhibition of DNA replication. Furthermore, transient transfection of an APC segment encoding amino acids 2140-2421 into a colon cancer cell line with mutant APC prevents cell cycle progression into or through S phase.


CONCLUSIONS
Our results suggest that APC can negatively regulate cell cycle progression through inhibition of DNA replication by direct interaction with DNA.
Qian
Jiang
J
Department of Molecular Virology, Immunology and Medical Genetics, Ohio State University, Columbus, 43210-2207, USA.
Sarnaik
Amod A
AA
Bonney
Tera M
TM
Keirsey
Jeremy
J
Combs
Kelly A
KA
Steigerwald
Kira
K
Acharya
Samir
S
Behbehani
Gregory K
GK
Barton
Michelle C
MC
Lowy
Andrew M
AM
Groden
Joanna
J
D025601:Adenomatous Polyposis Coli Protein / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000503:physiopathology; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D004261:DNA Replication / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016193:G1 Phase / Q000502:physiology; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D009865:Oocytes / Q000502:physiology; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D016196:S Phase / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology; D051153:Wnt Proteins / Q000378:metabolism; D014982:Xenopus laevis
D025601:Adenomatous Polyposis Coli Protein; D004268:DNA-Binding Proteins; D051153:Wnt Proteins; D018844:Cyclin-Dependent Kinases