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Analysis of Telomere Length in Specific Cells in Paraffin-Embedded Sections of Human Tissues
Telomeres are DNA sequences complexed with proteins located at the ends of each chromosome. In humans, a typical telomere DNA tract contains 1000 to 2000 tandem repeats of the sequence TTAGGG. Shortening of telomeres occurs at every cell division and may be caused by oxidative injury. Telomere activity increases telomere length and may immortalize cells in culture. Of particular interest is the finding that telomerase activity is detected in a large number of human cancers while it is not present in differentiated cells. Methods for the measurement of telomere length by fluorescent in situ hybridization (FISH) of metaphase chromosome spreads have been developed. Meeker et al (Am J Pathol 2002, 160:1259-1268) describe a method for quantitative assessment of telomere length which can be applied for formalin-fixed, paraffin-embedded human tissues. The assay requires only 10 to 15 cells and is adequate for small tissue samples and biopsies. Moreover, the telomere measuring technique can be combined with antibody detection by immunofluorescence, which permits the identification of specific cells for telomere analysis. Meeker and al used this novel technique to compare telomere lengths between tumor and normal epithelial cells in prostate sections.
Loss of DNA Mismatch Repair and Microsatellite Instability Is Similar in Adenomas and Carcinomas in Hereditary Nonpolyposis Colorectal Cancer
Germline mutations in DNA mismatch repair (MMR) loci are present in adenomas and colorectal carcinomas from individuals with hereditary nonpolyposis colorectal cancer (HNPCC). Loss of MMR causes microsatellite instability causing a large increase in mutation rats in simple DNA repeat sequences or at microsatellite loci. Deletions in long polyA repeats are sensitive indicators of microsatellite instability. Because the deletions take place in stepwise fashion, with loss of a single or only a few bases at a time, the extent of deletions in polyA repeats can provide information about the time interval since the loss of MMR. Kim et al (Am J Pathol 2002, 160:1503-1506) investigated at what time during tumor progression loss of MMR takes place. If loss of MMR occurs very early in tumorigenesis and may precede mutations in gatekeeper genes, the extent of deletions in polyA repeats should be similar between adenomas and carcinomas. Kim et al examined the extent of deletions in non-encoding polyA sequences in 6 adenomas and 10 cancers in HNPCC patients. Adenoma deletions were approximately the same as those in cancer. In addition, the extent of deletion in tumors removed from patients under clinical surveillance compared to non-surveillance patients did not differ significantly. The data demonstrate that microsatellite instability is quite extensive in both small adenomas and cancer. This finding is consistent with the notion that loss of MMR in HNPCC patients occurs very early in the tumorigenic process, probably before oncogene mutations.
Deficiency in DNA Mismatch Repair Enhances Endometrial Tumorigenesis Induced by Mutations in the PTEN Suppressor Gene
Mutations in the PTEN suppressor gene are the most common alterations in uterine endometrioid carcinoma (UEC). In addition, microsatellite instability resulting from defects of DNA mismatch repair occur in 20 to 45% of UEC. Both alterations are also detectable in complex atypical hyperplasia, the immediate precursor lesion for UEC. One hundred percent of mice lacking one wild-type copy of PTEN develop uterine complex atypical hyperplasia at 32 weeks and 20 to 25% of these mice develop invasive carcinoma at 40 weeks. Wang et al (Am J Pathol 2002, 160:1481-1486) generated double mutant mice which are heterozygous for PTEN and deficient in DNA mismatch repair (Meh1 nulls). By 14 to 18 weeks, the frequency of loss of the wild-type PTEN allele in the double mutant mice was as high as that present in lesions of 40-week-old PTEN heterozygous mice, and some of these mice already contained invasive lesions in the endometrium. The data demonstrate that deficiency of DNA mismatch repair accelerates endometrial tumorigenesis induced by PTEN mutations. The results also strongly suggest that the loss of the PTEN wild-type allele is an important step in the development of these tumors.
Detection of Skp2 (S-Phase Kinase-Associated Protein 2) as a Marker for Cell Replication in Lymphomas
Skp2 is a member of a family of proteins known as F-box proteins (Fbps) which share a 40 amino acid F-box motif. These proteins form complexes that can ubiquitinate a large number of proteins, marking them for proteasome degradation. Skp2 is required for the G1-S transition of the cell cycle and interacts with the cyclin A-Cdk2 complex. Skp2 deficient mice have slow growth while its overexpression in T cells causes oncogenesis in cooperation with ras genes. The cyclin-dependent kinase (CDK) inhibitor p27, which is often inactivated in human tumors, is a Skp2 substrate. Chiarle et al (Am J Pathol 2002, 160:1457-1466) demonstrate that Skp2 expression in lymphomas directly correlates with the grade of malignancy and has an inverse correlation with p27 expression in a majority of lymphoma subtypes. Although Skp2 is usually involved in p27 regulation, there are alternative pathways for regulation of this cell cycle inhibitor that may not involve Skp2. Nevertheless, Skp2 detection by flow cytometry or immunohistochemistry matched measurements of S-phase cells by DNA content, BrdU, and Ki-67 labeling. Detection of Skp2 offers a simple method to assess S-phase cells in lymphomas and may have prognostic value.
Nitric Oxide Protects Against Ischemia-Reperfusion Injury in Skeletal Muscle
Nitric oxide (NO) generated by the activity of endothelial NO synthase (eNOS) is an important regulator of vascular functions. Although NO is considered to be an important component of ischemia-reperfusion (I/R) tissue injury, it has not been established whether NO has a cytoprotective or cytotoxic role in the development of I/R injury. Studies with NO inhibitors have shown either protective or detrimental effects in this type of injury. Studies with transgenic mice, which express eNOS mostly in endothelium, showed that these animals are protected against several types of injury. Ozaki et al (Am J Pathol 2002, 160:1335-1344) investigated whether eNOS transgenic mice would be protected against I/R injury in skeletal muscle. During reperfusion, superoxide levels were decreased by approximately 50% in the transgenic mice compared to wild-type (WT) controls. In WT mice there were alterations in endothelial junctional proteins at the early stages of reperfusion as well as increased vascular permeability. In contrast, eNOS transgenic mice had no abnormalities in junctional proteins and no change in vascular permeability. Furthermore, in contrast to WT animals, leukocyte extravasation, increased expression of adhesion molecules, and decreased tissue viability were significantly inhibited in eNOS transgenics. This work demonstrates that eNOS overexpression and consequent NO production maintains vascular integrity and prevents I/R injury in skeletal muscle.
Angiopoietin 1 and 2 Antagonism in Tumor Neovascularization
Tumor cells can produce angiogenic factors which stimulate the
formation of new blood vessels required for tumor growth. In addition
to vascular endothelial growth factor (VEGF) and its receptors located
in endothelial cells, another system involving the endothelial tyrosine
kinase receptor, Tie2, appears to be important in vascular development.
Two ligands have been identified that bind the Tie2 receptor:
Angiopoietin1 (Ang1) which stimulates the receptor tyrosine kinase, and
Angiopoietin2 (Ang2) that acts as an antagonist for Ang1 by blocking
receptor phosphorylation. Tie2 activation by Ang1 is believed to
stabilize mature blood vessels. Ang2 is thought to block this effect
and facilitate the angiogenic response to VEGF. Although it is assumed
that the Ang1/Ang2 system may play an important role in tumor growth,
experimental results have not been consistent. Hawighorst et al
(Am J Pathol 2002, 160:1381-1392) analyzed the effects
of activation of the Tie2 receptor by Ang1 and Ang2 during mouse skin
carcinogenesis and in xenografts of squamous cell carcinomas.
Expression of Ang2 but not of Ang1 was increased in tumor blood vessels
even at the early stages of tumorigenesis. Stable overexpression of
Ang1 in tumor cells enhanced Tie2 phosphorylation and reduced tumor
growth. Blood vessels of these tumors had increased periendothelial
cells containing 
-smooth muscle actin, an indicator of vascular
maturity. The data demonstrate that during tumor growth there is an
angiogenic switch by which Ang2 counteracts the vessel-maturation
effects of Ang1.
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