Assessing the Toxoplasma Tachyzoite Cell Cycle Phases Using Fluorescent Ubiquitination-Based Cell Cycle Indicator.

Introduction High content screening (HCS) is a method that uses automatic microscopy and image analysis techniques to extract multiple phenotypically relevant measurements at cellular level. By now for high content screening there is a few technigues to evaluate cell cycle. This include BrdU staining and/or mitosis-specific marker phospho-histone H3 immunostaining. In our endeavors we used HeLa cells stably expressing FUCCI (Fluorescent Ubiquitination-based Cell-Cycle Indicator) probe. The FUCCI probe was generated by fusing red and green fluorescent proteins with ubiquitination domains of Cdt1 and Geminin respectively. As a means of tracking cell cycle progression FUCCI exploits cyclical expression and degradation of the ubiquitination oscillators Cdt1 and Geminin to specifically mark cell cycle phases in living cells. Cell cycle perturbation can be evaluated by measuring mean fluorescence intensity of Cdt1-Red and Geminin-Green in single cell and eventually based on fluorescence intensity range cells can be classified as one of G1, G1/S, S/G2/M and M cell cycle phase. The FUCCI technology can be used as alternative method for FACS and it is capable to procces more data than FACS. The goal was to set up robust assay to screen large compound collections to search for potential cell cycle modifiers. Brief description The assay was designed for full automatization on robotic station which has liquid dispenser, incubators and Yokogawa CV7000 microscope connected. This means that cell plating, addition of compounds, cytotoxicity measurement and high content microscopy is processed by robotic system. Images are exported to Columbus image analysis system and final results are processed in Tibco Spotfire software. For setting up and validation of the assay, dose response curve was generated for 8 reference compounds with known cell cycle activity. Summary of the new, unpublished data We have observed good assay reproducibility and robustness with Z’ value of above 0.5 for G1, S/G2/M and M phases. The assay methodology was used to test how commercially available LOPAC library, which includes 90 drug-like molecules from the fields of cell signaling and neuroscience. The results of cytotoxicity assay allowed to validate 26 compounds as active based on dose response curve fitting. For the first time FUCCI probe was used in high content screening experiments. The results demonstrates that several agents from LOPAC library (e.g. bosutinib, crizotinib, torcetrapib) were identified as cell cycle modulators. Statement of the conclusions The results we obtained shows that our robotic station is fully capable for primary screen for cell cycle modulators. This newly developed assay is simple to set up, robust, sensitive and inexpensive. Acknowledgement: Postup II CZ.1.07/2.3.00/30.0041 Citation Format: Pawel Znojek, Sona Gurska, Petr Dzubak, Marian Hajduch. Development and validation of high content screening assay for identification of compounds based on cytotoxicity and cell cycle analysis using FUCCI probe. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3692. doi:10.1158/1538-7445.AM2015-3692

The non-targeted effects of radiation have been known to induce significant alternations in cell survival. Although the effects might govern the progression of tumor sites following advanced radiotherapy, the impacts on the intercellular control of the cell cycle following radiation exposure with a modified field, remain to be determined. Recently, a fluorescent ubiquitination-based cell-cycle indicator (FUCCI), which can visualize the cell-cycle phases with fluorescence microscopy in real time, was developed for biological cell research. In this study, we investigated the non-targeted effects on the regulation of the cell cycle of human cervical carcinoma (HeLa) cells with imperfect p53 function that express the FUCCI (HeLa–FUCCI cells). The possible effects on the cell-cycle phases via soluble factors were analyzed following exposure to different field configurations, which were delivered using a 150 kVp X-ray irradiator. In addition, using synchrotron-generated, 5.35 keV monochromatic X-ray microbeams, high-precision 200 μm-slit microbeam irradiation was performed to investigate the possible impacts on the cell-cycle phases via cell–cell contacts. Collectively, we could not detect the intercellular regulation of the cell cycle in HeLa–FUCCI cells, which suggested that the unregulated cell growth was a malignant tumor. Our findings indicated that there was no significant intercellular control system of the cell cycle in malignant tumors during or after radiotherapy, highlighting the differences between normal tissue and tumor characteristics.

Cancer cells are methionine (MET) dependent compared to normal cells as they have an elevated requirement for MET in order to proliferate. MET restriction selectively traps cancer cells in the S/G2 phase of the cell cycle. The cell cycle phase can be visualized by color coding with the fluorescence ubiquitination-based cell cycle indicator (FUCCI). Recombinant methioninase (rMETase) is an enzyme that effectively degrades MET. rMETase induces S/G2-phase blockage of cancer cells which is identified by the cancer cells' green fluorescence with FUCCI imaging. Cancer cells in G1/G0 are the majority of the cells in solid tumors and are resistant to the chemotherapy. Treatment of cancer cells with standard chemotherapy drugs only led to the majority of the cancer cell population being arrested in G0/G1 phase, identified by the cancer cells' red fluorescence in the FUCCI system. The G0/G1-phase cancer cells are chemo-resistant. Tumor targeting Salmonella typhimurium A1-R (S. typhimurium A1-R) was used to decoy quiescent G0/G1 stomach cancer cells growing in nude mice to cycle, with subsequent rMETase treatment to selectively trap the decoyed cancer cells in S/G2 phase, which made them highly sensitive to chemotherapy. Subsequent cisplatinum (CDDP) or paclitaxel (PTX) chemotherapy was then administered to kill the decoyed and trapped cancer cells, which completely prevented or regressed tumor growth. In a subsequent experiment, a patient-derived orthotopic xenograft (PDOX) model of recurrent CDDP-resistant metastatic osteosarcoma was eradicated by the combination of Salmonella typhimurium A1-R decoy, rMETase S/G2-phase cell cycle trap, and CDDP cell kill. Salmonella typhimurium A1-R and rMETase pre-treatment thereby overcame CDDP resistance. These results demonstrate the effectiveness of the new chemotherapy paradigm of "decoy, trap, and kill" chemotherapy.

Cell survival after irradiation depends on the cell cycle at the time of exposure. This has been thought to be due to cell cycle-dependent nuclear DNA damage repair mechanisms. Here, we show the relationships between the exposed dose, the cell cycle phase at the time of exposure and changes in mitochondrial DNA copy numbers (mtDNAcn) after irradiation. We used a fluorescent ubiquitylation-based cell cycle indicator (FUCCI), which allows visualization of the cell cycle, and confirmed cell cycle synchronization in human cervical HeLa cells. In synchronous HeLa-FUCCI cells, the mtDNAcn changed with the progression of the cell cycle. Also, G1 phase-synchronized cells showed a dose-dependent increase of mtDNAcn at 48 h after X-ray exposure, whereas G2 cells showed a dose-dependent increase at 24 h. In addition, S phase-synchronized cells showed a dose-dependent increase at 24 and 48 h after irradiation. These results showed the cell cycle- and dose-dependent effects on mtDNAcn after irradiation, which might shed light on the emerging role of mitochondrial genome and in cell survival.

Effects of Proton Beams and X Rays on the Cell Cycle of Fluorescent Ubiquitination-Based Cell Cycle Indicator (Fucci)-Expressing Cells

We report here a mouse model of lymph node metastasis of stomach cancer and imaging cell cycle progression in the metastasis. FUCCI (fluorescent ubiquitination-based cell cycle indicator)-expressing human MKN45 gastric cancer cells were used to establish the lymph node metastasis nude mouse model. In this model, cell-cycle dynamics of the metastatic cells could be imaged in vivo. FUCCI cells are labeled orange in the G1 phase nuclei (monomeric Kusabira-Orange2: mKO2) and labeled green in the S/G2/M phases (monomeric Azami-Green1: mAG1). FUCCI MKN45 were injected into the mouse stomach mucosa from the serosal side. Lymph node metastasis to the para-aortic or hepatic portal region, left armpit, and brachial lymph nodes were observed. Green-yellow (G2/S) cells were predominant compared to red (G1) fluorescent FUCCI cells as observed in the lymph-node metastases using an FV1000 confocal microscope, indicating that the lymph-node metastatic cells were actively cycling. The origin of lymph node or vessel metastases was confirmed by H.E. staining. Mouse FUCCI gastric metastasis models will be used to visualize cell cycle dynamics in metastasis in lymphoid as well as hematogenous metastasis in order to optimize drug treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1694. doi:1538-7445.AM2012-1694

Apoptosis is induced by various stresses generated from the extracellular and intracellular environments. The fidelity of the cell cycle is monitored by surveillance mechanisms that arrest its further progression if any crucial process has not been completed or damages are sustained, and then the cells with problems undergo apoptosis. Although the molecular mechanisms involved in the regulation of the cell cycle and that of apoptosis have been elucidated, the links between them are not clear, especially that between cell cycle and death receptor-mediated apoptosis. By using the HeLa.S-Fucci (fluorescent ubiquitination-based cell cycle indicator) cells, we investigated the relationship between the cell cycle progression and apoptotic execution. To monitor apoptotic execution during cell cycle progression, we observed the cells after induction of apoptosis with time-lapse fluorescent microscopy. About 70% of Fas-mediated apoptotic cells were present at G1 phase and about 20% of cells died immediately after cytokinesis, whereas more than 60% of etoposide-induced apoptotic cells were at S/G2 phases in random culture of the cells. These results were confirmed by using synchronized culture of the cells. Furthermore, mitotic cells showed the resistance to Fas-mediated apoptosis. In conclusion, these findings suggest that apoptotic execution is dependent on cell cycle phase and Fas-mediated apoptosis preferentially occurs at G1 phase.

Curcumin (diferuloylmethane) is known to induce apoptosis in tumor cells. In asynchronous cultures, with time-lapse video-micrography in combination with quantitative fluorescence microscopy, we have demonstrated that curcumin induces apoptosis at G(2) phase of cell cycle in deregulated cyclin D1-expressed mammary epithelial carcinoma cells, leaving its normal counterpart unaffected. In our search toward delineating the molecular mechanisms behind such differential activities of curcumin, we found that it selectively increases p53 expression at G(2) phase of carcinoma cells and releases cytochrome c from mitochondria, which is an essential requirement for apoptosis. Further experiments using p53-null as well as dominant-negative and wild-type p53-transfected cells have established that curcumin induces apoptosis in carcinoma cells via a p53-dependent pathway. On the other hand, curcumin reversibly inhibits normal mammary epithelial cell cycle progression by down-regulating cyclin D1 expression and blocking its association with Cdk4/Cdk6 as well as by inhibiting phosphorylation and inactivation of retinoblastoma protein. In addition, curcumin significantly up-regulates cell cycle inhibitory protein (p21Waf-1) in normal cells and arrests them in G(0) phase of cell cycle. Therefore, these cells escape from curcumin-induced apoptosis at G(2) phase. Interestingly, these processes remain unaffected by curcumin in carcinoma cells where cyclin D1 expression is high. Similarly, in ectopically overexpressed system, curcumin cannot down-regulate cyclin D1 and thus block cell cycle progression. Hence, these cells progress into G(2) phase and undergo apoptosis. These observations together suggest that curcumin may have a possible therapeutic potential in breast cancer patients.

The cell cycle dependence of radiosensitivity has yet to be fully determined, as it is technically difficult to achieve a high degree of cell cycle synchronization in cultured cell systems and accurately detect the cell cycle phase of individual cells simultaneously. We used human cervical carcinoma HeLa cells expressing fluorescent ubiquitination-based cell cycle indicators (FUCCI), and employed the mitotic harvesting method that is one of the cell cycle synchronization methods. The imaging analysis confirmed that the cell cycle is highly synchronized after mitotic cell harvesting until 18-20 h of the doubling time has elapsed. Also, flow cytometry analysis revealed that the S and G2 phases peak at approximately 12 and 14-16 h, respectively, after mitotic harvesting. In addition, the clonogenic assay showed the changes in surviving fractions following exposure to X-rays according to the progress through the cell cycle. These results indicate that HeLa-FUCCI cells become radioresistant in the G1 phase, become radiosensitive in the early S phase, rapidly become radioresistant in the late S phase, and become radiosensitive again in the G2 phase. Our findings may contribute to the further development of combinations of radiation and cell cycle-specific anticancer agents.

Investigation of the relevance between cell cycle status and the bioactivity of exogenously delivered biomacromolecules is hindered by their time-consuming cell internalization and the cytotoxicity of transfection methods. In this study, we addressed these problems by utilizing the photochemical internalization (PCI) method using a peptide/protein-photosensitizer conjugate, which enables immediate cytoplasmic internalization of the bioactive peptides/proteins in a light-dependent manner with low cytotoxicity. To identify the cell-cycle dependent apoptosis, a TatBim peptide-photosensitizer conjugate (TatBim-PS) with apoptotic activity was photo-dependently internalized into HeLa cells expressing a fluorescent ubiquitination-based cell cycle indicator (Fucci2). Upon irradiation, cytoplasmic TatBim-PS internalization exceeded 95% for all cells classified in the G1, S, and G2/M cell cycle phases with no significant differences between groups. TatBim-PS-mediated apoptosis was more efficiently triggered by photoirradiation in the G1/S transition than in the G1 and S/G2/M phases, suggesting high sensitivity of the former phase to Bim-induced apoptosis. Thus, the cell cycle dependence of Bim peptide-induced apoptosis was successfully investigated using Fucci2 indicator and the PCI method. Since PCI-mediated cytoplasmic internalization of peptides is rapid and does not span multiple cell cycle phases, the Fucci-PCI method constitutes a promising tool for analyzing the cell cycle dependence of peptides/protein functions.

Mathematical Models for Cell Migration with Real-Time Cell Cycle Dynamics

Using an asynchronously growing cell population, we investigated how X-irradiation at different stages of the cell cycle influences individual cell–based kinetics. To visualize the cell-cycle phase, we employed the fluorescent ubiquitination-based cell cycle indicator (Fucci). After 5 Gy irradiation, HeLa cells no longer entered M phase in an order determined by their previous stage of the cell cycle, primarily because green phase (S and G2) was less prolonged in cells irradiated during the red phase (G1) than in those irradiated during the green phase. Furthermore, prolongation of the green phase in cells irradiated during the red phase gradually increased as the irradiation timing approached late G1 phase. The results revealed that endoreduplication rarely occurs in this cell line under the conditions we studied. We next established a method for classifying the green phase into early S, mid S, late S, and G2 phases at the time of irradiation, and then attempted to estimate the duration of G2 arrest based on certain assumptions. The value was the largest when cells were irradiated in mid or late S phase and the smallest when they were irradiated in G1 phase. In this study, by closely following individual cells irradiated at different cell-cycle phases, we revealed for the first time the unique cell-cycle kinetics in HeLa cells that follow irradiation.

Cell migration is a fundamental biological process of key importance in health and disease. Advances in imaging techniques have paved the way to monitor cell motility. An ever-growing collection of computational tools to track cells has improved our ability to analyze moving cells. One renowned goal in the field is to provide tools that track cell movement as comprehensively and automatically as possible. However, fully automated tracking over long intervals of time is challenged by dividing cells, thus calling for a combination of automated and supervised tracking. Furthermore, after the emergence of various experimental tools to monitor cell-cycle phases, it is of relevance to integrate the monitoring of cell-cycle phases and motility. We developed CellMAPtracer, a multiplatform tracking system that achieves that goal. It can be operated as a conventional, automated tracking tool of single cells in numerous imaging applications. However, CellMAPtracer also allows adjusting tracked cells in a semiautomated supervised fashion, thereby improving the accuracy and facilitating the long-term tracking of migratory and dividing cells. CellMAPtracer is available with a user-friendly graphical interface and does not require any coding or programming skills. CellMAPtracer is compatible with two- and three-color fluorescent ubiquitination-based cell-cycle indicator (FUCCI) systems and allows the user to accurately monitor various migration parameters throughout the cell cycle, thus having great potential to facilitate new discoveries in cell biology.

Uncovering Cell Cycle-Dependent Effects on Cell Survival in Near-Infrared Photoimmunotherapy.

Osteoporosis affects over 20 million patients in the United States. Among those, disuse osteoporosis is serious as it is induced by bed-ridden conditions in patients suffering from aging-associated diseases including cardiovascular, neurological, and malignant neoplastic diseases. Although the phenomenon that loss of mechanical stress such as bed-ridden condition reduces bone mass is clear, molecular bases for the disuse osteoporosis are still incompletely understood. In disuse osteoporosis model, bone loss is interfered by inhibitors of sympathetic tone and adrenergic receptors that suppress bone formation. However, how beta adrenergic stimulation affects osteoblastic migration and associated proliferation is not known. Here we introduced a live imaging system, fluorescent ubiquitination-based cell cycle indicator (FUCCI), in osteoblast biology and examined isoproterenol regulation of cell cycle transition and cell migration in osteoblasts. Isoproterenol treatment suppresses the levels of first entry peak of quiescent osteoblastic cells into cell cycle phase by shifting from G1 /G0 to S/G2 /M and also suppresses the levels of second major peak population that enters into S/G2 /M. The isoproterenol regulation of osteoblastic cell cycle transition is associated with isoproterenol suppression on the velocity of migration. This isoproterenol regulation of migration velocity is cell cycle phase specific as it suppresses migration velocity of osteoblasts in G1 phase but not in G1 /S nor in G2 /M phase. Finally, these observations on isoproterenol regulation of osteoblastic migration and cell cycle transition are opposite to the PTH actions in osteoblasts. In summary, we discovered that sympathetic tone regulates osteoblastic migration in association with cell cycle transition by using FUCCI system.