Dataset Information

Cell-cycle fate-monitoring distinguishes individual chemosensitive and chemoresistant cancer cells in drug-treated heterogeneous populations demonstrated by real-time FUCCI imaging.

ABSTRACT: Essentially every population of cancer cells within a tumor is heterogeneous, especially with regard to chemosensitivity and resistance. In the present study, we utilized the fluorescence ubiquitination-based cell cycle indicator (FUCCI) imaging system to investigate the correlation between cell-cycle behavior and apoptosis after treatment of cancer cells with chemotherapeutic drugs. HeLa cells expressing FUCCI were treated with doxorubicin (DOX) (5 ?M) or cisplatinum (CDDP) (5 ?M) for 3 h. Cell-cycle progression and apoptosis were monitored by time-lapse FUCCI imaging for 72 h. Time-lapse FUCCI imaging demonstrated that both DOX and CDDP could induce cell cycle arrest in S/G2/M in almost all the cells, but a subpopulation of the cells could escape the block and undergo mitosis. The subpopulation which went through mitosis subsequently underwent apoptosis, while the cells arrested in S/G2/M survived. The present results demonstrate that chemoresistant cells can be readily identified in a heterogeneous population of cancer cells by S/G2/M arrest, which can serve in future studies as a visible target for novel agents that kill cell-cycle-arrested cells.

SUBMITTER: Miwa S

PROVIDER: S-EPMC4613186 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Cell-cycle fate-monitoring distinguishes individual chemosensitive and chemoresistant cancer cells in drug-treated heterogeneous populations demonstrated by real-time FUCCI imaging.

Miwa Shinji S Yano Shuya S Kimura Hiroaki H Yamamoto Mako M Toneri Makoto M Matsumoto Yasunori Y Uehara Fuminari F Hiroshima Yukihiko Y Murakami Takashi T Hayashi Katsuhiro K Yamamoto Norio N Bouvet Michael M Fujiwara Toshiyoshi T Tsuchiya Hiroyuki H Hoffman Robert M RM

Cell cycle (Georgetown, Tex.) 20150101 4

Essentially every population of cancer cells within a tumor is heterogeneous, especially with regard to chemosensitivity and resistance. In the present study, we utilized the fluorescence ubiquitination-based cell cycle indicator (FUCCI) imaging system to investigate the correlation between cell-cycle behavior and apoptosis after treatment of cancer cells with chemotherapeutic drugs. HeLa cells expressing FUCCI were treated with doxorubicin (DOX) (5 μM) or cisplatinum (CDDP) (5 μM) for 3 h. Cell ...[more]

PMID: 25551170

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Project description:Ovarian cancer ranks eighth in cancer incidence and mortality among women worldwide. Cisplatin-based chemotherapy is commonly used for patients with ovarian cancer. However, the clinical efficacy of cisplatin is limited due to the occurrence of adverse side effects and development of cancer chemoresistance during treatment. Trans-(±)-kusunokinin has been previously reported to inhibit cell proliferation and induce cell apoptosis in various cancer cell types, including breast, colon and cholangiocarcinoma. However, the potential effects of (±)-kusunokinin on ovarian cancer remains unknown. In the present study, chemosensitive ovarian cancer cell line A2780 and chemoresistant ovarian cancer cell lines A2780cis, SKOV-3 and OVCAR-3 were treated with trans-(±)-kusunokinin to investigate its potential effects. MTT, colony formation, apoptosis and multi-caspase assays were used to determine cytotoxicity, the ability of single cells to form colonies, induction of apoptosis and multi-caspase activity, respectively. Moreover, western blot analysis was performed to determine the proteins level of topoisomerase II, cyclin D1, CDK1, Bax and p53-upregulated modulator of apoptosis (PUMA). The results demonstrated that trans-(±)-kusunokinin exhibited the strongest cytotoxicity against A2780cis cells with an IC50 value of 3.4 µM whilst also reducing the colony formation of A2780 and A2780cis cells. Trans-(±)-kusunokinin also induced the cells to undergo apoptosis and increased multi-caspase activity in A2780 and A2780cis cells. This compound significantly downregulated topoisomerase II, cyclin D1 and CDK1 expression, but upregulated Bax and PUMA expression in both A2780 and A2780cis cells. In conclusion, trans-(±)-kusunokinin suppressed ovarian cancer cells through the inhibition of colony formation, cell proliferation and the induction of apoptosis. This pure compound could be a potential targeted therapy for ovarian cancer treatment in the future. However, studies in an animal model and clinical trial need to be performed to support the efficacy and safety of this new treatment.

Project description:The Core Binding Factor (CBF) protein RUNX1 is a master regulator of definitive hematopoiesis, crucial for hematopoietic stem cell (HSC) emergence during ontogeny. RUNX1 also plays vital roles in adult mice, in regulating the correct specification of numerous blood lineages. Akin to the other mammalian Runx genes, Runx1 has two promoters P1 (distal) and P2 (proximal) which generate distinct protein isoforms. The activities and specific relevance of these two promoters in adult hematopoiesis remain to be fully elucidated. Utilizing a dual reporter mouse model we demonstrate that the distal P1 promoter is broadly active in adult hematopoietic stem and progenitor cell (HSPC) populations. By contrast the activity of the proximal P2 promoter is more restricted and its upregulation, in both the immature Lineage- Sca1high cKithigh (LSK) and bipotential Pre-Megakaryocytic/Erythroid Progenitor (PreMegE) populations, coincides with a loss of erythroid (Ery) specification. Accordingly the PreMegE population can be prospectively separated into "pro-erythroid" and "pro-megakaryocyte" populations based on Runx1 P2 activity. Comparative gene expression analyses between Runx1 P2+ and P2- populations indicated that levels of CD34 expression could substitute for P2 activity to distinguish these two cell populations in wild type (WT) bone marrow (BM). Prospective isolation of these two populations will enable the further investigation of molecular mechanisms involved in megakaryocytic/erythroid (Mk/Ery) cell fate decisions. Having characterized the extensive activity of P1, we utilized a P1-GFP homozygous mouse model to analyze the impact of the complete absence of Runx1 P1 expression in adult mice and observed strong defects in the T cell lineage. Finally, we investigated how the leukemic fusion protein AML1-ETO9a might influence Runx1 promoter usage. Short-term AML1-ETO9a induction in BM resulted in preferential P2 upregulation, suggesting its expression may be important to establish a pre-leukemic environment.

Project description:RATIONALE:Understanding and manipulating the cardiomyocyte cell cycle has been the focus of decades of research, however the ultimate goal of activating mitotic activity in adult mammalian cardiomyocytes remains elusive and controversial. The relentless pursuit of controlling cardiomyocyte mitosis has been complicated and obfuscated by a multitude of indices used as evidence of cardiomyocyte cell cycle activity that lack clear identification of cardiomyocyte "proliferation" versus cell cycle progression, endoreplication, endomitosis, and even DNA damage. Unambiguous appreciation of the complexity of cardiomyocyte replication that avoids oversimplification and misinterpretation is desperately needed. OBJECTIVE:Track cardiomyocyte cell cycle activity and authenticate fidelity of proliferation markers as indicators of de novo cardiomyogenesis in post-mitotic cardiomyocytes. METHODS AND RESULTS:Cardiomyocytes expressing the FUCCI construct driven by the α-myosin heavy chain promoter were readily and uniformly detected through the myocardium of transgenic mice. Cardiomyocyte cell cycle activity peaks at postnatal day 2 and rapidly declines thereafter with almost all cardiomyocytes arrested at the G1/S cell cycle transition. Myocardial infarction injury in adult hearts prompts transient small increases in myocytes progressing through cell cycle without concurrent mitotic activity, indicating lack of cardiomyogenesis. In comparison, cardiomyogenic activity during early postnatal development correlated with coincidence of FUCCI and cKit+ cells that were undetectable in the adult myocardium. CONCLUSIONS:Cardiomyocyte-specific expression of Fluorescence Ubiquitination-based Cell Cycle Indicators (FUCCI) reveals previously unappreciated aspects of cardiomyocyte cell cycle arrest and biological activity in postnatal development and in response to pathologic damage. Compared to many other methods and model systems, the FUCCI transgenic (FUCCI-Tg) mouse represents a valuable tool to unambiguously track cell cycle and proliferation of the entire cardiomyocyte population in the adult murine heart. FUCCI-Tg provides a desperately needed novel approach in the armamentarium of tools to validate cardiomyocyte proliferative activity that will reveal cell cycle progression, discriminate between cycle progression, DNA replication, and proliferation, and provide important insight for enhancing cardiomyocyte proliferation in the context of adult myocardial tissue.

Dataset Information

Cell-cycle fate-monitoring distinguishes individual chemosensitive and chemoresistant cancer cells in drug-treated heterogeneous populations demonstrated by real-time FUCCI imaging.

Publications

Cell-cycle fate-monitoring distinguishes individual chemosensitive and chemoresistant cancer cells in drug-treated heterogeneous populations demonstrated by real-time FUCCI imaging.

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