Menno F. Kielman

Mutations in the APC tumour suppressor gene cause chromosomal instability

Two forms of genetic instability have been described in colorectal cancer: microsatellite instability and chromosomal instability. Microsatellite instability results from mutations in mismatch repair genes; chromosomal instability is the hallmark of many colorectal cancers, although it is not completely understood at the molecular level. As truncations of the Adenomatous Polyposis Coli (APC) gene are found in most colorectal tumours, we thought that mutations in APC might be responsible for chromosomal instability. To test this hypothesis, we examined mouse embryonic stem (ES) cells homozygous for Min (multiple intestinal neoplasia) or Apc1638T alleles. Here we show that Apc mutant ES cells display extensive chromosome and spindle aberrations, providing genetic evidence for a role of APC in chromosome segregation. Consistent with this, APC accumulates at the kinetochore during mitosis. Apc mutant cells form mitotic spindles with an abundance of microtubules that inefficiently connect with kinetochores. This phenotype is recapitulated by the induced expression of a 253-amino-acid carboxy-terminal fragment of APC in microsatellite unstable colorectal cancer cells. We conclude that loss of APC sequences that lie C-terminal to the β-catenin regulatory domain contributes to chromosomal instability in colorectal cancer.

Apc modulates embryonic stem-cell differentiation by controlling the dosage of β-catenin signaling

The Wnt signal-transduction pathway induces the nuclear translocation of membrane-bound β-catenin (Catnb) and has a key role in cell-fate determination. Tight somatic regulation of this signal is essential, as uncontrolled nuclear accumulation of β-catenin can cause developmental defects and tumorigenesis in the adult organism. The adenomatous polyposis coli gene (APC) is a major controller of the Wnt pathway and is essential to prevent tumorigenesis in a variety of tissues and organs. Here, we have investigated the effect of different mutations in Apc on the differentiation potential of mouse embryonic stem (ES) cells. We provide genetic and molecular evidence that the ability and sensitivity of ES cells to differentiate into the three germ layers is inhibited by increased doses of β-catenin by specific Apc mutations. These range from a severe differentiation blockade in Apc alleles completely deficient in β-catenin regulation to more specific neuroectodermal, dorsal mesodermal and endodermal defects in more hypomorphic alleles. Accordingly, a targeted oncogenic mutation in Catnb also affects the differentiation potential of ES cells. Expression profiling of wildtype and Apc-mutated teratomas supports the differentiation defects at the molecular level and pinpoints a large number of downstream structural and regulating genes. Chimeric experiments showed that this effect is cell-autonomous. Our results imply that constitutive activation of the Apc/β-catenin signaling pathway results in differentiation defects in tissue homeostasis, and possibly underlies tumorigenesis in the colon and other self-renewing tissues.

SomaticApc mutations are selected upon their capacity to inactivate the ?-catenin downregulating activity

The APC gene, originally identified as the gene for familial adenomatous polyposis (FAP), is now considered as the true “gatekeeper” of colonic epithelial proliferation. Its main tumor suppressing activity seems to reside in the capacity to properly regulate intracellular β‐catenin signaling. Most somatic APC mutations are detected between codons 1286 and 1513, the mutation cluster region (MCR). This clustering can be explained either by the presence of mutation‐prone sequences within the MCR, or by the selective advantage provided by the resulting truncated polypeptides. Here, a Msh2‐deficient mouse model (Msh2Δ7N ) was generated and bred with Apc1638N and ApcMin that allowed the comparison of the somatic mutation spectra along the Apc gene in the different allelic combinations. Mutations identified in Msh2Δ7N/Δ7N tumors are predominantly dinucleotide deletions at simple sequence repeats leading to truncated Apc polypeptides that partially retain the 20 a.a. β‐catenin downregulating motifs. In contrast, the somatic mutations identified in the wild type Apc allele of Msh2Δ7N/Δ7N /Apc+/1638N and Msh2Δ7N/Δ7N /Apc+/Min tumors are clustered more to the 5′ end, thereby completely inactivating the β‐catenin downregulating activity of APC. These results indicate that somatic Apc mutations are selected during intestinal tumorigenesis and that inactivation of the β‐catenin downregulating function of APC is likely to represent the main selective factor.

A targeted mouse Brca1 mutation removing the last BRCT repeat results in apoptosis and embryonic lethality at the headfold stage.

A mouse model with a targeted mutation in the 3′ end of the endogenous Brca1 gene, Brca11700T, was generated to compare the phenotypic consequences of truncated Brca1 proteins with other mutant Brca1 models reported in the literature to date. Mice heterozygous for the Brca11700T mutation do not show any predisposition to tumorigenesis. Treatment of these mice with ionizing radiation or breeding with Apc, Msh-2 or Tp53 mutant mouse models did not show any change in the tumor phenotype. Like other Brca1 mouse models, the Brca11700T mutation is embryonic lethal in homozygous state. However, homozygous Brca11700T embryos reach the headfold stage but are delayed in their development and fail to turn. Thus, in contrast to Brca1null models, the mutant embryos do not undergo growth arrest leading to a developmental block at 6.5 dpc, but continue to proliferate and differentiate until 9.5 dpc. Homozygous embryos die between 9.5–10.5 dpc due to massive apoptosis throughout the embryo. These results indicate that a C-terminal truncating Brca1 mutation removing the last BRCT repeat has a different effect on normal cell function than does the complete absence of Brca1.

Dynamic expression and nuclear accumulation of β-catenin during the development of hair follicle-derived structures

Beta-catenin has a dual role in the cell. At the membrane, it connects E-cadherin to the actin cytoskeleton, while in the nucleus, it controls gene expression in concert with Tcf-like transcription factors. Nuclear translocation of beta-catenin is induced by the Wnt signal transduction pathway. Control of this process is essential since elevated beta-catenin levels interfere with differentiation and development, and can initiate cancer in many tissues. An important role for beta-catenin during hair follicle related development and tumorigenesis has recently been established, though little is known of its endogenous expression during the development of these structures. Here, we have investigated the expression of beta-catenin in relation to markers for proliferation, differentiation and Wnt signaling during the development of three hair follicle related structures, i.e. whiskers, normal body hair and the preputial gland, and a hair follicle-derived tumor, the epidermal cyst. We observed nuclear accumulation of beta-catenin, the hallmark of Wnt signaling, in the upper matrix, the dermal papilla, the developing ringwulst of the whisker and in the tumor, though it was never in association with proliferation or terminal differentiation. Co-localization of nuclear beta-catenin with Tcf-3/4 was found only in the dermal papilla and the developing ringwulst of the whisker, but not in the upper matrix or in the tumor. These results further elucidate the role of the Wnt signal transduction pathway during hair follicle related development and tumorigenesis and illustrate the dynamic role of beta-catenin in signal transduction and cell-adhesion.

Genetic polymorphism of the major regulatory element HS-40 upstream of the human α-globin gene cluster

Summary. The highly conserved 350‐bp major regulatory element HS‐40 (or αMRE) upstream of the human α‐globin gene cluster is involved in the regulation of α‐globin gene expression. The study of αMRE differences between human populations and the evolution of αMRE sequences in mammals may lead to a better understanding of the function and importance of this element in the regulation of expression of the downstream α‐cluster. Denaturing gradient gel electrophoresis was used to determine the sequence heterogeneity of the αMRE region in 276 unrelated individuals, representing seven different populations. Furthermore, we analysed the α major regulatory elements of chimpanzee, orang‐utan and rhesus monkeys and compared them with the equivalent human and murine sequences. Six different αMRE haplotypes (labelled A to F) were found in humans. Haplotype frequencies between the seven populations showed a gradual shift to a higher haplotype A distribution from west to east, being the highest in Indonesians. The African sample shows the largest divergence in haplotypes. Five out of six different haplotypes were present, three of which were exclusively found in Africans. The high prevalence of the haplotype A in humans, together with the conservation of this haplotype in apes, suggests that it is the ancestral one. The αMRE fragment appears to be a highly polymorphic marker, which could be used in combination with the regular markers in the α‐cluster to extend the haplotype and to follow segregation of α‐thalassaemia genes in population studies more accurately.

Erratum: Apc modulates embryonic stem-cell differentiation by controlling the dosage of β-catenin signaling (Nature Genetics (2002) 32 (594-605))

Nature Genet. 32, 650–654 (2002). Published online 11 November 2002; doi:10.1038/ng1047 A typographical error in the third sentence of the abstract resulted in the P value for the association being incorrectly reported as P = 0.00000033. The correct value is, in fact, P = 0.0000033.