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Methylator Phenotype: A Mechanism for Colorectal Carcinogenesis
Hypermethylation of multiple islands of the sequence CpG is a newly described mechanism in the development of colorectal carcinomas and adenomas. The CpG methylator phenotype is generally referred to as CIMP (CpG island methylator phenotype). Chan et al (Am J Pathol 2002, 160:529–536) studied CIMP in patients with multiple hyperplastic polyps (HP), large HPs, or hyperplastic polyposis. Methylation at several loci was analyzed by methylation-specific polymerase chain reaction. Sporadic HPs were not methylated at any locus. In contrast, 43% of HPs from hyperplastic polyposis or large/multiple polyps had two or more methylated sites. High CIMP was present in patients with predominance of HPs in right colon and was associated with absence of K-ras mutations. Detection of concordant CpG island methylation in some types of HP support the notion that some patients may have a hypermethylator phenotype in colorectal lesions. However, it has not been established whether the hypermethylator phenotype is associated with genetic predisposition or carcinogen exposure.
ß-Catenin Alterations in Colorectal Carcinogenesis: A Review
Many studies have demonstrated the dysregulation of ß-catenin pathways in colorectal carcinogenesis and the association between ß-catenin and APC protein. However, there are multiple mechanisms involved in carcinogenesis that may be associated with ß-catenin pathways. Many factors can contribute to the release of ß- catenin from the plasma membrane and its nuclear translocation. Moreover, the association of nuclear ß-catenin with TCF/LEF transcription factors enhances the expression of a large number of genes, including cell-cycle genes and COX-2. Wong and Pignatelli (Am J Pathol 2002, 160:389–401) review these mechanisms, discuss their possible contribution to colorectal carcinogenesis and emphasize that ß-catenin is a key participant in carcinogenesis.
Focal Proteolysis during Angiogenesis: Shedding of MMP Vesicles by Endothelial Cells
During normal and pathological angiogenesis endothelial cells produce metalloproteases (MMP) that are highly regulated at the synthesis and activation levels. Taraboletti et al (Am J Pathol 2002, 160:673–680) investigated whether MMPs may be secreted by endothelial cells in culture. They demonstrate that human umbilical vein endothelial cells (HUVEC) shed vesicles from the plasma membrane containing two gelatinases, MMP-2 and MMP-9, in active and proenzyme forms, the MT1-MMP proenzyme and the inhibitors TIMP-1 and TIMP-2. Shedding of vesicles is a rapid process stimulated by exposure to serum or the angiogenic factors FGF2 and vascular endothelium growth factor (VEGF). HUVEC exposed to the shed vesicles became invasive on a Matrigel assay. The authors suggest that shedding of MMP-containing vesicles by endothelial cells induces focal proteolysis essential for angiogenesis.
Induction of VEGF Production Causes Retinal Neovascularization and Detachment
Overexpression of vascular endothelium growth factor (VEGF) induces neovascularization in the retina with invasion into the subretinal space. Ohno-Matsui et al (Am J Pathol 2002, 160:711–719) established lines of transgenic mice in which VEGF expression driven by a rhodopsin or an interphotoreceptor retinoid binding protein (IRBP) is inducible. VEGF inducibility was achieved by the use of a tetracycline gene transactivator that responds to doxycycline. Induction of VEGF expression resulted in extensive retinal neovascularization within 3 to 4 days leading to total retinal detachment. This work demonstrates that in adult animals, the inducible expression of VEGF causes severe neovascularization and detachment of the retina as a consequence of traction produced by outer retinal folds. This animal model should have wide applicability in studies of ocular neovascularization.
Stathmin Expression May Be Required for Maintenance of Axonal Integrity
Stathmin is a highly conserved protein that is abundantly expressed in central and peripheral nervous systems. Surprisingly, however, no apparent abnormalities have been described in knock-out mice that are deficient in stathmin. Liedtke et al (Am J Pathol 2002, 160:469–480) examined aging stathmin knock-out mice and report that these animals have axonal lesions in both the central and peripheral nervous system. Advanced lesions included axon degeneration, dysmyelination, and a special type of glial reaction. Aging stathmin knock-out mice had reduced motor nerve conduction velocity and increased expression of the SCG 10-like protein, a stathmin-related gene that destabilizes microtubules. The authors conclude that stathmin expression may be essential to maintain axon integrity in aging animals and that its absence is associated with increased expression of axonal destabilizing proteins.
Ethanol Fixation Preserves Tissues for Molecular Analysis
One of the major difficulties in the molecular analysis of human tissue samples is to establish methods for tissue fixation that do not damage RNA, DNA, and proteins for subsequent study. The usual method of tissue preparation from tissue specimens is formalin fixation followed by paraffin embedding. However, it is known that this method causes extensive protein cross-linking and is also not suitable for use in cDNA microarray technology. Gillespie et al (Am J Pathol 2002, 160:449–457) report that fixation of tissues in 70% ethanol followed by paraffin embedding provides satisfactory results for molecular analyses performed by one- and two-dimensional gel electrophoresis as well as layered expression scanning and immunoblotting. Although mRNA recovery was reduced, ethanol fixation proved to be superior to formalin-fixed tissues for the performance of polymerase chain reaction and for DNA recovery. The authors conclude that 70% ethanol fixation and paraffin-embedded tissues retain their morphological features and can be used for analyses performed by sensitive molecular techniques.
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