To benefit from the fluorescence-based automatic microscope (FLAME), we have adapted a PNA FISH technique to automatically determine telomere length in interphase nuclei. The method relies on the simultaneous acquisition of pan-telomeric signals and reference probe signals. We compared the quantitative figures to those for existing methods, i.e. Southern blot analysis and quantitative FISH (Q-FISH). Quantitative-FISH on interphase nuclei (IQ-FISH) allows the exact quantification of telomere length in interphase nuclei. Thus, this enables us to obtain not only exact information on the telomere length, but also morphological and topological details. The automatic measurement of large cell numbers allows the measurement of statistically relevant cell populations. Key terms: automatic; quantification; telomere; FISH; microscopy; fluorescence; ALT; senescence Monitoring of telomere length regulation has become an important aspect of stem-cell research, studies on cellular senescence, and cancer research. A number of techniques have been developed to study telomere length in a quantitative manner. The standard procedure is Southern blot hybridization (1). The quantitative fluorescence in situ hybridization (Q-FISH) on metaphase chromosomes allows the quantification of the telomeric FISH signals on individual chromosomes (2-4). Since fluorescence intensity of the telomeric signals was found to be proportional to the size of telomeric repeats, Q-FISH is now widely used (2). For measuring telomere length in interphase nuclei, flow cytometry or fluorescence microscopy can be applied (5-7). Nonadhesive hematopoietic cells seem to be better suited for flow cytometric analyses than solid tumor cells (8,9). Therefore, for the analysis of nonhematopoietic cells, interphase FISH is the method of choice. However, the fluorescence microscopical method to quantify telomere length in interphase nuclei has so far only been performed manually on a restricted number of cells (6,7). To combine the positive aspects of flow cytometric measurements with the ability to quantify individual nuclei by fluorescence microscopical examination, we took advantage of fluorescence-based automatic microscope (FLAME). Thus, all the advantages of interphase measurements, i.e., the analysis of individual cells and the applicability to nonproliferating cells, can be combined with the analyses of statistically relevant cell populations. To develop a reliable method to automatically quantify telomere length in interphase nuclei, we applied a two-color hybridization assay and measured the fluorescence signals with FLAME. We then described the parameters essential for intra-and interexperimental comparisons. We performed intensity measurements in interphase nuclei and compared the results of single channel measurements of the target probe with the results obtained after introducing an internal reference and performing double channel measurements. We validated the quantitative-FISH on interphase nuclei (IQ-FISH) method by measuring telomere lengths of differe...

Recently, it was shown that MYCN amplified cells spontaneously expulse extrachromosomally amplified gene copies by micronuclei formation. Furthermore, it was shown that these cells lose their malignant phenotype and start to age. We tested whether it is possible to encourage neuroblastoma tumor cells to enter the senescence pathway by low concentrations of the micronuclei-inducing drug hydroxyurea (HU). We studied the effect of HU on 12 neuroblastoma cell lines with extra- or intrachromosomally amplified MYCN copies and without amplification. Two extrachromosomally amplified neuroblastoma cell lines (with double minutes) were investigated in detail. Already after 3 weeks of HU treatment, the BrdU uptake dropped to 25% of the starting cells. After 4 weeks, enlarged and flattened cells (F-cells) and increased granularity in the majority of cells were observed. A drastic reduction of the MYCN copy number-down to one copy per cell-associated with CD44 and MHCI upregulation in up to 100% of the HU treated neuroblastoma cells was found after 5-8 weeks. Telomere length was reduced to half the length within 8 weeks of HU treatment, and telomerase activity was not detectable at this time, while being strongly expressed at the beginning. All these features and the expression of senescence-associated-beta-galactosidase (SA-beta-GAL) in up to 100% of the cells support the hypothesis that these cells entered the senescence pathway. Thus, low-dose HU is a potent senescence elicitor for tumor cells with gene amplification, possibly representing an attractive additional strategy for treatment of this subset of tumors.

Background: The reliable detection and quantification of gene amplifications is crucial to clinical practice. Although there are different detection techniques, the fluorescence in situ hybridization (FISH) method has become highly accepted over past years because it is a reliable, robust, and quick method. Unfortunately, automatic quantification of gene amplification based on fluorescence intensities has not been possible thus far. Because current spot counting methods are reliable only when analyzing low amplification rates, we attempted to establish another method, i.e., to quantify the intensity of different FISH signals using an automatic fluorescence microscopical device on interphase nuclei: interphase quantitative FISH (IQ-FISH).Methods: We quantified the fluorescence intensities of the differently labeled FISH probes (MYCN and D2Z) hybridized to three different neuroblastoma cell lines, six peripheral blood (PB) samples, 10 spiked PB samples, and nine neuroblastoma samples using the Metafer4 system (MetaSystems, Altlussheim, Germany). To obtain the MYCN copy number per cell, the ratio between the fluorescence intensities of the MYCN gene and reference sequence (D2Z) was calculated. For automatic analysis of the HER-2/neu status in tumor cells, labeled FISH probes specific for HER-2/neu and a chromosome 17-specific probe were hybridized to peripheral blood and tumor specimens and analyzed using the automatic device. Results: When measuring the fluorescence intensity per cell for both probe pairs (MYCN/D2Z and HER-2/17p), amplified and non-amplified cells, showed distinct peaks with only little overlap. Whereas normal cells showed a fluorescence ratio peak for MYCN/D2Z between 200 and 800, cells with MYCN amplification clearly exceeded this ratio value (1000 to 25,000). When mixing a varying number of MYCNamplified cells (range 9 -91%) to normal PB, the spiked tumor cells could be identified. Even one neuroblastoma tumor cell in 1000 mononucleated cells could reliably be detected using our device. In neuroblastoma patient samples, non-amplified cells were distinguished from amplified cells. Automatically and manually counted signals gave matching results in amplified and non-amplified samples. HER-2/neuamplified cells were automatically detected in the breast cancer samples analyzed.Conclusion: The automatic measurement of fluorescence signal intensities not only allows a reliable discrimination between non-amplified and amplified cells but also exact quantification of amplified sequences. This is the prerequisite for the following applications: detection of amplified cells in the bone marrow and second-look specimens; comparison between primary and relapse or pre-and post-chemotherapeutic specimens; detection of tumors with focal gene amplification; and quantification of elimination of amplified gene sequences.