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• W2132610552 abstract "The concept of cells moving during proliferation through discrete intermittent steps of the “cell cycle” until the nuclear DNA has been duplicated and separates into two individual cell nuclei has its 60th anniversary. The cell cycle phases G1, S, M, and G2 that are now common knowledge to all students of biomedical sciences had been coined mid-last century by Alma Howard and Stephen P. Pelc in their groundbreaking but slightly forgotten work 1. They developed new technologies to understand DNA synthesis using autoradiography and fostered new ways to “sort” different cell cycle phases by improved gradient centrifugation, both cutting edge technologies at their time. In the following more than half a century, the model of the cell cycle was extremely useful to understand the complex and intricate ways that cells control their DNA replication, DNA damage repair, and cell elimination and how dysregulations may lead to diseases like cancer. Numerous regulatory molecules were discovered and their acting in concert at the various checkpoints in space and time was identified. In these discoveries flow cytometry was an important driving force. It enabled to clearly identify and count cells in different cell cycle phases, allowed investigation of cell cycle phase, specifically expression and interaction of regulatory molecules, and finally, their isolation. Image cytometry further contributed to the gain of knowledge by quantitative imaging and analysis of morphological changes and spatiotemporal interrelations. But it is still an open question why, in contrast to normal cells, cell cycling of neoplastic cells is dysregulated. Early on, a restriction point was proposed that regulates cell cycle in the G1-phase, for example under starvation conditions. However, this was so-far commonly demonstrated for cultured but not for primary cells. Now, Jiang and colleagues from Wuhan, China (this issue, page 944) scrutinized the cell cycle progression of PHA stimulated primary human peripheral blood lymphocytes. The authors used different flow cytometry assays to test for cell cycle phase, DNA synthesis, Cyclin E, P21 and Ki-67 expression, and apoptosis. They show evidence that also in primary cells the restriction point exists and that it is possibly not exclusive for cells in the G1-phase. To follow the subcellular and nuclear processes, methods with high resolution are necessary to analyze several cell properties in combination, in order to yield multiparametric information directed and analyzed by highly sophisticated software algorithms. Imaging flow cytometry analysis has the high information content of microscopy such as morphology and can be combined with quantitative properties of multiparametric cytometry. So it was assessed quantitatively that p65 translocation in immunophenotypically defined subpopulations is highly reproducible. NF-KB translocation in leukemic cell lines correlated well with microscopic and Western blot analysis and the Daunorubicin-induced biological response correlated quantitatively with the nuclear translocation of NF-KB 2. Manual evaluation of fluorescence microscopy images is an extremely time-consuming task with relatively low reproducibility. However, up to now robust algorithms were developed for routine analysis and accurate segmentation of microscopic images of different cell types by automated image processing 3. Intracellular nucleic acid and protein distributions across a population of Chinese hamster ovary (CHO-K1) cells have been directly mapped using imaging spectrophotometry with paired images in the 200- to 280-nm wavelength range 4. It also became possible to determine differences in repair kinetics between various cell types by the use of imaging flow cytometry. This approach has also permitted an evaluation of foci in a large number of cells (20,000) for different cell lines 5. Another important question is how replication and DNA damage repair are topologically and causally related to each other in the nucleus. Histone γH2AX proved to be very important in DNA repair processes as revealed by “click-chemistry” 6, 7. In earlier studies, these authors investigated the effects of oxidative stress 7 and topoisomerase inhibitors 8 on DNA-damage signaling and reviewed their relationship to the cell cycle 9. Quantitative confocal microscopic analysis of DNA damage and replication processes gave an innovative opportunity to scrutinize its chronological and spatial correlation with other nuclear processes in greater detail. To this end, Berniak and colleagues and Bernas and colleagues from Krakow and Warsaw, Poland, and New York, USA (this issue, page 913 and page 925, respectively) developed and improved new 3D image analysis methods to investigate the spatial interrelationship of DNA damage (histone γH2AX foci) and DNA replication sites (EdU incorporation) in relation to the DNA content (i.e., the cells' position in the cell cycle). For the nuclei (DAPI) γH2AX (AlexaFluor 568) and EdU (AlexaFluor 488) of a human lung carcinoma cell line 3D images were created and X,Y,Z coordinates of the centers of mass of the fluorescence spots were calculated. Nearest distances within individual nuclei were determined between coordinates of the γH2AX foci and the EdU sites. It was found that the distribution was cell cycle phase dependent and that the late S-phase appeared to be the most suitable phase for assessing a coincidence between DNA damage and DNA replication. The 3D images generated from γH2AX and EdU foci are not only scientifically informative but also an aesthetically interesting eye-opener. Spontaneous, i.e. not external agent-induced, DNA damage may also have relevance in disease development. Obesity, a highly relevant and emerging civilizational disease, harbors a strong risk for inducing physiological changes that may lead to cardiovascular, skeletal, or hormonal diseases. For studying human fat cell biology in cell culture, adipocytes from patients with Simpson-Golabi-Behmel Syndrome (SGBS cells) have proved to be a useful model 10. The gold standard for apoptosis confirmation is the cell's morphology and today's demand is the unbiased automated analysis of this process. SGBS cells were used by Doan-Xuan and colleagues from Debrecen, Hungary, and Ulm, Germany (this issue, page 933) for detailed high-content image cytometry investigation of the development and death of cultured human adipocytes. They used Nile blue and Nile red staining and changes in light absorption to differentiate preadipocyte–adipocyte development during the adipogenesis process. By their developed multicolor cytometric method, the classical apoptotic features which include cell shrinkage, nuclear condensation, internuclosomal DNA cleavage, and preadipocyte–adipocyte development as well as apoptosis induction could be detected. For apoptosis quantitation, a special preparative technique for passive diffusion of fragmented DNA from the cell nucleus was developed and applied using Low Melting Point agarose-covered cell cultures in combination with alkaline lysis buffer treatment. This method affects the chromatin and the cell nucleus containing fragmented DNA, showing an extra DNA layer “halo” around the nucleus. Specimens were quantified by laser scanning cytometry. Apoptotic cells could then be identified both by phosphatidylserine externalization detected by Annexin V staining and the ratio of halo and central region. The central region contains the majority of the unfragmented DNA but the outside layer that contains fragmented DNA is increased in apoptotic cells as compared to normal adipocytes. These examples highlight how important multiparametric flow and image cytometry are to unravel processes throughout cells' life during “cycling,” damage repair, and death. In addition, image cytometry is of utmost relevance for the detailed investigations of processes that involve changes in the cells' morphology as well as location and kinetics of sub-cellular structures." @default.
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• W2132610552 date "2013-09-20" @default.
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• W2132610552 title "DNA amplification and repair: Further insights by cytometry" @default.
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• W2132610552 doi "https://doi.org/10.1002/cyto.a.22392" @default.
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