قياس حركية تقدم دورة الخلية مع وصفها الأيض والتدفق الخلوي
Summary
ويمكن تحقيق بعض التغييرات الطفيفة في تتبع تطور وحركية من مراحل دورة الخلية عن طريق استخدام مزيج من العلامات الأيضية من الأحماض النووية مع BrdU ومجموع تلطيخ الحمض النووي الجيني عن طريق يوديد Propidium. هذا الأسلوب يتجنب الحاجة لتزامن الكيميائية من الخلايا ركوب الدراجات، وبالتالي منع إدخال الحمض النووي من التلف غير محددة، وهذا بدوره يؤثر على تقدم دورة الخلية.
Abstract
Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism.
The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments.
Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging.
Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.
Protocol
Discussion
من خلال الجمع بين التدفق الخلوي مع دمج BrdU، لدينا الأدوات اللازمة لدراسة حركية دورة الخلية. الخاصية المميزة للBrdU في أداء وظيفته التماثلية ثيميدين هو ما يسمح لتقدير محتوى الحمض النووي للخلية وركوب الدراجات. إدماج BrdU إلى حبلا ابنة الحمض النووي للنمو خلال مرحلة التوليف …
Divulgations
The authors have nothing to disclose.
Acknowledgements
نشكر اندي جونسون من مركز البحوث الطبية الحيوية في جامعة كولومبيا البريطانية للحصول على المساعدة مع تحليل نظام مراقبة الأصول الميدانية. يتم توفير التمويل للبحوث السرطانية في المختبر وونغ من قبل المعهد الكندي لابحاث السرطان في المجتمع (منحة التشغيل # 019250)، وإعادة الاستثمار من أموال البحوث التابع لكلية العلوم الصيدلية، جامعة كولومبيا البريطانية. ويدعم JMYW من كراسي البحث وكندا، ومايكل سميث مؤسسة لبرامج الصحة بحوث التطوير الوظيفي.
Materials
| Reagent | Company | Catalogue Number | Comments |
| bromodeoxyuridine | Becton Dickinson | 55089 | |
| propidium iodide | Sigma | 287075 | 1mg/ml stock |
| FITC anti-BrdU | Becton Dickinson | 347583 | |
| sodium tetraborate | Fisher | S80172 | 0.1M, pH 8.5 |
| FACS Caliber | Becton Dickinson |
References
- Musgrove, E. A., Caldon, C. E., Barraclough, J., Stone, A., Sutherland, R. L. Cyclin D as a therapeutic target in cancer. Nat. Rev. Cancer. 11, 558-572 (2011).
- Molchadsky, A., Rivlin, N., Brosh, R., Rotter, V., Sarig, R. p53 is balancing development, differentiation and de-differentiation to assure cancer prevention. Carcinogenesis. 31, 1501-1508 (2010).
- Dickson, M. A., Schwartz, G. K. Development of cell-cycle inhibitors for cancer therapy. Curr. Oncol. 16, 36-43 (2009).
- Banfalvi, G. Cell cycle synchronization of animal cells and nuclei by centrifugal elutriation. Nat. Protoc. 3, 663-673 (2008).
- van Opstal, A., Boonstra, J. Inhibitors of phosphatidylinositol 3-kinase activity prevent cell cycle progression and induce apoptosis at the M/G1 transition in CHO cells. Cell Mol. Life Sci. 63, 220-228 (2006).
- Gasnereau, I. Flow cytometry to sort mammalian cells in cytokinesis. Cytometry. A. 71, 1-7 (2007).
- Harper, J. V. Synchronization of cell populations in G1/S and G2/M phases of the cell cycle. Methods Mol. Biol. 296, 157-166 (2005).
- Pedrali-Noy, G. Synchronization of HeLa cell cultures by inhibition of DNA polymerase alpha with aphidicolin. Nucleic Acids Res. 8, 377-387 (1980).
- Merrill, G. F. Cell synchronization. Methods Cell Biol. 57, 229-249 (1998).
- Fleisig, H. B., Wong, J. M. Telomerase promotes efficient cell cycle kinetics and confers growth advantage to telomerase-negative transformed human cells. Oncogene. , (2011).
- Cai, D., Byth, K. F., Shapiro, G. I. AZ703, an imidazo[1,2-a]pyridine inhibitor of cyclin-dependent kinases 1 and 2, induces E2F-1-dependent apoptosis enhanced by depletion of cyclin-dependent kinase 9. Cancer Res. 66, 435-444 (2006).
- Pozarowski, P., Darzynkiewicz, Z. Analysis of cell cycle by flow cytometry. Methods Mol. Biol. 281, 301-311 (2004).
- Sherwood, S. W., Rush, D. F., Kung, A. L., Schimke, R. T. Cyclin B1 expression in HeLa S3 cells studied by flow cytometry. Exp. Cell Res. 211, 275-281 (1994).
