HUMAN tumorigenesis is associated with the accumulation of mutations both in oncogenes and in tumour suppressor genes1–3 But in no common adult cancer have the mutations that are critical in the early stages of the tumorigenic process been defined We have attempted to determine if mutations of the APC gene play such a role in human colorectal tumours, which evolve from small benign tumours (adenomas) to larger malignant tumours (carcinomas) over the course of several decades Here we report that sequence analysis of 41 colorectal tumours revealed that the majority of colorectal carcinomas (60%) and adenomas (63%) contained a mutated APC gene Furthermore, the APC gene met two criteria of importance for tumour initiation First, mutations of this gene were found in the earliest tumours that could be analysed, including adenomas as small as 05 cm in diameter Second, the frequency of such mutations remained constant as tumours progressed from benign to malignant stages These data provide strong evidence that mutations of the APC gene play a major role in the early development of colorectal neoplasms

APC mutations occur early during colorectal tumorigenesis.

Mutations of the genes encoding APC or beta-catenin in colon carcinoma induce the constitutive formation of nuclear beta-catenin/Tcf-4 complexes, resulting in activated transcription of Tcf target genes. To study the physiological role of Tcf-4 (which is encoded by the Tcf7/2 gene), we disrupted Tcf7/2 by homologous recombination. Tcf7/2-/- mice die shortly after birth. A single histopathological abnormality was observed. An apparently normal transition of intestinal endoderm into epithelium occurred at approximately embryonic day (E) 14.5. However, no proliferative compartments were maintained in the prospective crypt regions between the villi. As a consequence, the neonatal epithelium was composed entirely of differentiated, non-dividing villus cells. We conclude that the genetic program controlled by Tcf-4 maintains the crypt stem cells of the small intestine. The constitutive activity of Tcf-4 in APC-deficient human epithelial cells may contribute to their malignant transformation by maintaining stem-cell characteristics.

Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4.

Caught up in a Wnt storm: Wnt signaling in cancer.

Neurofibromatosis type 2 (NF2) is a monogenic dominantly inherited disease predisposing carriers to develop nervous system tumours. To identify the genetic defect, the region between two flanking polymorphic markers on chromosome 22 was cloned and several genes identified. One is the site of germ-line mutations in NF2 patients and of somatic mutations in NF2-related tumours. Its deduced product has homology with proteins at the plasma membrane and cytoskeleton interface, a previously unknown site of action of tumour suppressor genes in humans.

Alteration in a new gene encoding a putative membrane-organizing protein causes neuro-fibromatosis type 2

To assess the critical role of Wnt signals in intestinal crypts, we generated transgenic mice ectopically expressing Dickkopf1 (Dkk1), a secreted Wnt inhibitor. We find that epithelial proliferation is greatly reduced coincidentally with the loss of crypts. Although enterocyte differentiation appears unaffected, secretory cell lineages are largely absent. Disrupted intestinal homeostasis is reflected by an absence of nuclear β-catenin, inhibition of c-myc expression, and subsequent up-regulation of p21CIP1/WAF1. Thus, our data are the first to establish a direct requirement for Wnt ligands in driving proliferation in the intestinal epithelium, and also define an unexpected role for Wnts in controlling secretory cell differentiation.

/pdf/canonical-wnt-signals-are-essential-for-homeostasis-of-the-4uwoyl76p1.pdf

Canonical Wnt signals are essential for homeostasis of the intestinal epithelium

Germ-line mutations in the human adenomatous polyposis coli (APC) gene result in familial adenomatous polyposis, an autosomal dominant disorder characterized by the early onset of multiple adenomatous polyps in the large bowel with a high likelihood of developing colorectal carcinomas. To understand the role of APC in intestinal tumor formation, we have introduced a chain-termination mutation in the 15th exon of the mouse Apc gene and employed it to modify the endogenous gene by homologous recombination in embryonic stem cells. Mice which are heterozygous for the Apc gene modification progressively develop intestinal tumors in a manner that is similar to that observed in patients with familial adenomatous polyposis and in mice which carry a mutation called multiple intestinal neoplasia (Min). Our results indicate that the Apc gene modification is a critical event in the initiation of intestinal tumor formation and results in an autosomal dominant predisposition toward development of spontaneous colonic and intestinal tumors in mice.

https://www.pnas.org/content/pnas/91/19/8969.full.pdf

A targeted chain-termination mutation in the mouse Apc gene results in multiple intestinal tumors

Maltose/proton co-transport was studied in intact cells and in plasma membrane vesicles of the yeast Saccharomyces cerevisiae. In order to determine uphill transport in vesicles, plasma membranes were fused with proteoliposomes containing cytochrome c oxidase as a proton-motive force-generating system. Maltose accumulation, dependent on the electrical and pH gradients, was observed. The initial uptake velocity and accumulation ratio in vesicles proved to be dependent on the external pH. Moreover, kinetic analysis of maltose transport showed that Vmax. values greatly decreased with increasing pH, whereas the Km remained virtually constant. These observations were in good agreement with results obtained with intact cells, and suggest that proton binding to the carrier proceeds with an apparent pK of 5.7. The observation with intact cells that maltose is co-transported with protons in a one-to-one stoichiometry was ascertained in the vesicle system by measuring the balance between proton-motive force and the chemical maltose gradient. These results show that maltose transport in vesicles prepared by fusion of plasma membranes with cytochrome c oxidase proteoliposomes behaves in a similar way as in intact cells. It is therefore concluded that this vesicle model system offers a wide range of new possibilities for the study of maltose/proton co-transport in more detail.

/pdf/maltose-proton-co-transport-in-saccharomyces-cerevisiae-rha92w44pw.pdf

Maltose/proton co-transport in Saccharomyces cerevisiae. Comparative study with cells and plasma membrane vesicles.

/pdf/ca-repeat-polymorphism-at-the-d5s299-locus-linked-to-3s1m6q77iw.pdf

C. van Leeuwen

Papers

A targeted chain-termination mutation in the mouse Apc gene results in multiple intestinal tumors

Maltose/proton co-transport in Saccharomyces cerevisiae. Comparative study with cells and plasma membrane vesicles.

CA repeat polymorphism at the D5S299 locus linked to adenomatous polyposis coli (APC).

CA repeat polymorphism at the D5S82 locus, proximal to adenomatous polyposis coli (APC).

Polyphosphate synthesis in yeast