Survivin-induced Aurora-B kinase activation: A mechanism by which APC mutations contribute to increased mitoses during colon cancer development.

APC mutations initiate most colorectal cancers (CRCs), but cellular mechanisms linking this to CRC pathology are unclear. We reported that wild-type APC in the colon down-regulates the anti-apoptotic protein survivin, and APC mutation up-regulates it, explaining why most CRCs display survivin overexpression and apoptosis inhibition. However, it does not explain another hallmark of CRC pathology--increased mitotic figures and cell proliferation. Because survivin activates aurora-B kinase (ABK) in vitro, catalyzing mitosis, we hypothesized that in normal colonic crypts, APC controls ABK activity, while in neoplastic APC-mutant crypts, ABK activity is up-regulated, increasing mitosis. We quantitatively mapped intracryptal distributions of survivin, ABK, and markers of activated downstream signaling and mitosis (INCENP, phospho-histone-H3, phospho-centromere-protein-A). In normal crypts, gradients for these markers, ABK:survivin:INCENP complexes, and ABK activity were highest in the lower crypt (inverse to the APC gradient). In neoplastic crypts that harbor APC mutations, proliferating (Ki-67+) cells and cells expressing survivin, ABK, and phospho-histone-H3 were distributed farther up the crypt. Hence, as cells migrate up neoplastic crypts, transitions between cell phenotypes (eg, from stem to proliferating) appear delayed. In CRC cell lines, increasing wild-type APC, inhibiting TCF-4, or decreasing survivin expression down-regulated ABK activity. Thus, APC mutation-induced up-regulation of the survivin/ABK cascade can explain delayed crypt cell maturation, expansion of proliferative cell populations (including mitotic figures), and promotion of colon tumorigenesis.

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.

Improvement in sensitivity of allele-specific PCR facilitates reliable noninvasive prenatal detection of cystic fibrosis.

BACKGROUND: Cell-free fetal DNA circulating in maternal blood has potential as a safer alternative to invasive methods of prenatal testing for paternally inherited genetic alterations, such as cystic fibrosis (CF) mutations. METHODS: We used allele-specific PCR to detect mutated CF D1152H DNA in the presence of an excess of the corresponding wild-type sequence. Pfx buffer (Invitrogen) containing replication accessory proteins and Taq polymerase with no proofreading activity was combined with TaqMaster PCR Enhancer (Eppendorf) to suppress nonspecific amplification of the wild-type allele. The procedure was tested on DNA isolated from plasma drawn from 11 pregnant women (gestational age, 11-19.2 weeks), with mutation confirmation by chorionic villus sampling. RESULTS: The method detected 5 copies of the CF D1152H mutant allele in the presence of up to approximately 100,000 copies of wild-type allele without interference from the wild-type sequence. The D1152H mutation was correctly identified in one positive sample; the only false-positive result was seen in a mishandled sample. CONCLUSIONS: This procedure allows for reliable detection of the paternally inherited D1152H mutation and has potential application for detection of other mutations, which may help reduce the need for invasive testing.