A Tcf4-GFP reporter mouse model for monitoring effects of Apc mutations during intestinal tumorigenesis.
Apc mutations cause intestinal tumorigenesis through Tcf4 activation. However, direct techniques for studying Tcf4 activation in vivo are limited. Here, we describe the development of a Tcf4-GFP reporter mouse model for directly studying Tcf4 activation. We first developed a GFP reporter construct (Tcf4-GFP) and transfected it into SW480 cells that have constitutively activated Tcf4. Reporter activity increased 47-fold. Next, we created transgenic (Tg) mice by transducing the construct into C57BL/6J mice. Fluorescence microscopy did not detect GFP in intestinal sections, but flow cytometry showed 5% of crypt cells to be GFP(+). We then established cross-bred mice (Tg x Apc(Min/+)), which have a germline Apc mutation and sustained Tcf4 activation. Here, fluorescence microscopy showed GFP(+) cells at or near the base of normal-appearing crypts. In adenomas, in which Apc is inactivated, GFP(+) signal was even greater. Immunostaining for the Tcf4 target genes survivin (BIRC5) and cyclin D1 (CCND1) showed that their expression also paralleled GFP positivity. We conclude that GFP directly reports Tcf4 activation in vivo and tracks the predicted increases in Tcf4 activation that result from Apc inactivation, and that Apc mutation contributes to survivin and cyclin D1 overexpression through Tcf4 activation. Our Tcf4 mouse should be useful in studying the effects of chemopreventive agents on Wnt signaling and changes in proliferative crypt cell populations-including stem cells-during intestinal tumorigenesis.
Boman
Bruce
B
Thomas Jefferson University, Kimmel Cancer Center, Philadelphia, Pennsylvania, USA.
Kopelovich
Levy
L
Siracusa
Linda D
LD
Zhang
Tao
T
Henderson
Kim
K
Cofer
Zenobia
Z
Buchberg
Arthur M
AM
Fields
Jeremy Z
JZ
Otevrel
Tomas
T
D000236:Adenoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D025601:Adenomatous Polyposis Coli Protein / Q000235:genetics / Q000502:physiology*; D000818:Animals; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D045744:Cell Line, Tumor; D019938:Cyclin D1 / Q000378:metabolism; D020895:Enterocytes / Q000378:metabolism / Q000473:pathology; D005260:Female; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D051034:Inhibitor of Apoptosis Proteins; D007414:Intestinal Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D019394:Ki-67 Antigen / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins; D000077022:Survivin; D051820:TCF Transcription Factors / Q000235:genetics / Q000378:metabolism*; D000073940:Transcription Factor 4
D025601:Adenomatous Polyposis Coli Protein; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; C117749:Birc5 protein, mouse; D051034:Inhibitor of Apoptosis Proteins; D019394:Ki-67 Antigen; D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D000077022:Survivin; D051820:TCF Transcription Factors; C090743:Tcf4 protein, mouse; D000073940:Transcription Factor 4; D019938:Cyclin D1; D049452:Green Fluorescent Proteins; D008156:Luciferases