ACF And Tumorigenesis In The Colon Of DMH-Treated Rat

Objective Aberrant crypt foci (ACF) were described in 1987 by Bird and Good as putative preneoplastic lesions in the colon of carcinogen- treated rodents. ACF are defined by their characteristic morphology: the crypts are enlarged, they have thickened layer of epithelial cells, they have increased pericryptal space, they have irregular lumens, and they are microscopically elevated. ACF are identified in histologic sections and by scanning electron microscopy. ACF have been used as a short-term bioassay to evaluate the role of nutritional components and , chemopreventive agents at an early stage of colon carcinogenesis . However, although they are thought to be the earliest identifiable neoplasitic lesions in the colon carcinogenetic model., the development of these lesions is not clearly related to the early development of tumors. Therefore, there is a strong need to clarify the role of ACF in colon carcinogenesis and to validate their relevance as biomarkers of tumorigenesis.The Wnt pathway controls cell fate during embryonic development, it not only drives tumorigenesis but is also required at different stages of gut development. Wnts activate responding cells by interacting with the seven-span transmembrane protein Frizzled (Fz) and the single-span transmembrane protein LRP. Two functional complexes involving these proteins have been described. Wnts may simultaneously bind to Fz and LRP. which leads to the formation of nuclear Tcf/β-catenin complexes. The key component of the Wnt canonical cascade is the cytoplasmic proteinβ-catenin. Subsequently, glycogen synthase kinase 3β(GSK3β) is recruited to phosphorylate additional serine and threonine residues N-terminal to S45. Together, these proteins make up the so-calledβ-catenin destruction complex. In the presence of Wnt stimulation, theβ-catenin destruction complex is destabilized, andβ-catenin accumulates in the nucleus to activate transcription of Tcf target genes (c-myc,cylinD1,,MMP-7,etal).β-catenin may be regarded as existing in three different subcellular forms: membrane-bound (as part of the adherens complex), cytosolic, and nuclear. Binding of the protein to other members of the adherens complex, ie, E-cadherin andβ-catenin, is thought to be regulated by tyrosine phosphorylation. Tyrosine phosphorylation ofβ-catenin leads to its dissociation from the adherens complex10 and probable transfer of the protein to the cytosol where it exists in a soluble, monomeric state.Cytosolicβ-catenin may subsequently be degraded or be translocated into the nucleus. The degradation ofβ-catenin involves binding of the protein to a complex involving APC protein, and two further proteins, AXIN and glycogen synthase kinase (GSK)-3β.The latter serves to phosphorylate serine and threonine residues onβ-catenin, a crucial step required to target the protein for ubiquitination and proteosomal degradation. Both APC and AXIN enhance this phosphorylation and are, therefore,promoters ofβ-catenin degradation.As the target gene of wnt/β-catenin signal way, MMP-7 is one member family of matrilysin. MMP-7 (matrilysin) is expressed in up to 90% of CRCs and is thought to be important in mediating stromal invasion, but may also have a role in earlier stages of carcinogenesis. In support of the latter, ablation of MMP-7 expression reduces the number of intestinal adenomas in APCmin/ mice. Co-localization of nuclearβ-catenin and MMP-7 protein and mRNA has been demonstrated in human CRCs and tumors from APCmin/mice, respectively. The human MMP-7 gene promoter has at least two TCF-binding sites but, unlike with COX-2, theβ-catenin /TCF complex has been shown to increase MMP-7 promoter activity in vitro. Further, this effect is abrogated by mutating a TCF-binding site on the MMP-7 promoter or by introducing a dominant-negative TCF mutant.Now we evaluated the relationship between ACF and tumorigenesis by observing the sequential development from ACF to tumor in DMH-treated rats.to evaluate the change of the proliferation and the apoptosis in the different stage of tumorgenesis in the colon of rat's model ,and to valuate the significance ofβ-catenin and MMP-7 expression in ACF and tumor with immunohistochemical method.methods1. 60 Wistar rats (s.c.DMH 15mg·kg^-1 bw×18) were killed sequentially from weeks 8 to 25 after the first DMH injection ,the number of rats killed at each time point after DMH treatment is:wk8-14, wk16-21,wk22-25. the colons stained with methylene blue were transilluminated in the stereomicroscopy . the changes of lesions in morphology were observed through hematoxylin-eosin (HE) staining.2.Expression ofβ-catenin and MMP-7 was detected with immunohistochemical method in ACF and tumors,to explore the signal way that the histogenesis of colorectal carcinoma.Result1. There are two types of early lesions: classic ACF and dark ACF.dark ACF were characterized by dark blue staining,mild enlarged crypts, compressed crypt openings.Dark ACF and nascent tumors displayed the same surface morphology,Furthermore,they grew significantly faster than classic ACF,and showed dysplasia without hyperplasia. In contrast, classic ACF showed hyperplasia but never dysplasia..2. There was significantly difference between classical ACF and dark ACF about the expression rate ofβ-catenin and MMP-7. The adnormal expressed rate ofβ-catenin is 4.84% and 100%(P=0.000), respectively. The MMP-7 expression rate is 7.87% and 81.82% (P=0.000) .There was no significantly difference between dark ACF and tumor about the expression rate ofβ—catenin and MMP-7.3. The distribution of most ACF is between 50 and 90%. In total, 13 tumors developed in rats, three were seen in the proximal 10% and 20%. Histology showed that all of them were poorly differentiated, mucin-secreting carcinomas . ten tumors were found in the distal colon beyond the 50% point. Histology showed three to be adenomas and seven to be well-differentiated adenocarcinomas .Conclusion1. Not all of the ACF related with tumor, in the colon of Wistar rats exposed toDMH, we identified two kinds of ACF: dark ACF and classic ACF. Dark ACF displayed a continuous development from early stages to tumor with their characteristic morphology, fast crypt multiplication and the same expression ofβ-catenin and MMP-7. In contrast, classic ACF do not seem to be as closely related to tumorigenesis.2. The cytoplasmic and nuclearβ-catenin expression , probably an early event, was related to the development of dark ACF transform to adenoma and colorectal cancer.β-catenin may enhance the degradative function of the target gene MMP-7 through nuclear translocationand may further facilitate dark ACF transform to colorectal tumor. The increasing of Expression ofβ-catenin and MMP-7 result in Wnt signal way's activation in ACF and tumor ,it means ACF and tumor come from the same signal way.3. Our study suggested the distribution of ACF and adenomas and well-differentiated adenocarcinomas is same.