Pathology and genetics of tumours of the urinary system and male genital organs

It has long been presumed impossible to measure telomeres in vertebrate DNA by PCR amplification with oligonucleotide primers designed to hybridize to the TTAGGG and CCCTAA repeats, because only primer dimer-derived products are expected. Here we present a primer pair that eliminates this problem, allowing simple and rapid measurement of telomeres in a closed tube, fluorescence-based assay. This assay will facilitate investigations of the biology of telomeres and the roles they play in the molecular pathophysiology of diseases and aging.

Telomere measurement by quantitative PCR

https://delangelab.rockefeller.edu/pubs/50_Griffith_Cell_99.pdf

Mammalian Telomeres End in a Large Duplex Loop

Telomere Shortening and Tumor Formation by Mouse Cells Lacking Telomerase RNA

Switching and Signaling at the Telomere

Fiber FISH as a DNA Mapping Tool

SUMMARY

The application of light microscopy in medicine and cell biology has been significantly influenced by both the availability of specific biological reagents such as monoclonal antibodies and nucleic acid probes, as well as by the enormous progress in microelectronics and computer technology. It is likely that specific reagents for a variety of cellular macromolecules will become available on a large scale in the coming years. Moreover, methods using both sensitive detection devices such as charge-coupled device (CCD) cameras and sophisticated image processing exist to quantify this information at the single molecule level in morphologically intact cells.



This paper describes the impact of these two factors on the light microscope of the future, with special emphasis on fluorescence. It defines the improvements that still are required to solve some of the challenging problems such as the quantification of unique genes and their products in intact cells, the quantification of DNA adducts and the detection of rare mutant cells or circulating tumour cells.

Does light microscopy have a future

The detection of rare-event cells circulating in peripheral blood using automated image analysis was evaluated using a model system consisting of cells from a breast cancer cell line (SKBR3) seeded in a mononuclear cell suspension. Slides of cells with optimal morphology were prepared according to an optimized preparation procedure based on centrifugal cytology in combination with formalin fixation. SKBR3 cells were immunocytochemically stained for cytokeratin using the cam 5.2 monoclonal antibody and labelled with alkaline phosphatase using CAS-red as substrate. Because, for optimal segmentation of cell images, plain differences in absorption wavelength are required, the red immunostaining was combined with a green nuclear counter-staining based on ethyl green. Slides were automatically screened for cytokeratin-positive SKBR3 cells resulting in a lowest detectable frequency of one positive cell per 1.87 x 10(6) negative cells. A comparison between manual screening and automated screening for cytokeratin-positive cells showed a high level of correlation (0.9998). For the definition of the total number of objects per slide, two counting procedures were evaluated. Results were close to the visual score with a coefficient of variation of 0.47% for the counting procedure used in this study. It is concluded that optimization of preparation and staining procedures for the detection of rare-event cells using automated image analysis results in optimal image contrast and, consequently, in an increase in sensitivity for detecting rare events.

Detection of immunocytochemically stained rare events using image analysis

The expression of the adenosine deaminase complexing protein (ADCP) was investigated by immunohistochemistry in the normal and hyperplastic human prostate, in 30 prostatic adenocarcinomas, and in seven human prostatic adenocarcinoma cell lines grown as xenografts in athymic nude mice. In the normal and hyperplastic prostate, ADCP was localized exclusively in the apical membrane and the apical cytoplasm of the glandular epithelial cells. In prostatic adenocarcinomas, four distinct ADCP expression patterns were observed: diffuse cytoplasmic, membranous, both cytoplasmic and membranous, and no ADCP expression. The expression patterns were compared with the presence of metastases. We found an inverse correlation between membranous ADCP immunoreactivity and metastatic propensity. Exclusively membranous ADCP immunoreactivity occurred only in non-metastatic tumours. In contrast, the metastatic tumours showed no or diffuse cytoplasmic ADCP immunoreactivity. This suggests that immunohistochemical detection of ADCP might predict the biological behaviour of prostatic cancer. However, the occurrence of membranous ADCP immunoreactivity in the xenograft of a cell line (PC-EW), derived from a prostatic carcinoma metastasis, indicates that not only the tendency to metastasize modulates ADCP expression.

Hans J. Tanke

Papers

Fiber FISH as a DNA Mapping Tool

Does light microscopy have a future

Detection of immunocytochemically stained rare events using image analysis

Adenosine deaminase complexing protein (ADCP) expression and metastatic potential in prostatic adenocarcinomas

Exon mapping by fiber-FISH or LR-PCR.