Epithelial-to-Mesenchymal Transition Promotes Tubulin Detyrosination and Microtentacles that Enhance Endothelial Engagement

Whipple, Rebecca A.; Matrone, Michael A.; Cho, Edward H.; Balzer, Eric M.; Vítolo, Michele; Yoon, Jennifer R.; Ioffe, Olga B.; Tuttle, Kimberly C.; Yang, Jing; Martin, Stuart S.

doi:10.1158/0008-5472.can-09-4613

Cited by 127 publications

(138 citation statements)

References 47 publications

(71 reference statements)

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“…In this study, we used an impedanciometry technique to obtain qualitative information in real-time on the biological status of CCRF-CEM cells, including degree of adhesion, spreading and proliferation. PBOX-15 reduced the Cell Index of CCRF-CEM cells compared to control cells within 1 h, indicating that this effect on the phenotype of the cells was independent of cell cycle arrest and subsequent apoptosis, and suggesting that PBOX-15 rapidly inhibited the adhesion and spreading of the cells (34,35). Of note is the more pronounced and sustained effect of PBOX-15 on the cells compared to treatment with nocodazole ( Fig.…”

Section: Discussionmentioning

confidence: 79%

The microtubule targeting agent PBOX-15 inhibits integrin-mediated cell adhesion and induces apoptosis in acute lymphoblastic leukaemia cells

Lysaght

Verma

Maginn

et al. 2012

International Journal of Oncology

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Abstract. Although recent decades have seen an improved cure rate for newly diagnosed paediatric acute lymphoplastic leukaemia (ALL), the treatment options for adult ALL, T-cell ALL (T-ALL) and relapsed disease remain poor. We have developed a novel series of pyrrolo-1,5-benzoxazepine (PBOX) compounds and established their anticancer efficacy in a variety of human tumour cell types. Here, we demonstrate that PBOX-15 inhibits cell growth, and induces G2/M cell cycle arrest and apoptosis in both T-ALL and B-cell ALL (B-ALL) cells. In addition, prior to PBOX-15-induced apoptosis, PBOX-15 decreases ALL cell adhesion, spreading and migration. Concurrently, PBOX-15 differentially down-regulates β1-, β2-and α4-integrin expression in ALL cells and significantly decreases integrin-mediated cell attachment. PBOX-15 interferes with the lateral mobility and clustering of integrins in both B-ALL and T-ALL cells. These data suggest that PBOX-15 is not only effective in inducing apoptosis in ALL cells, but also has the potential to disrupt integrin-mediated adhesion of malignant lymphocytes, which represents a novel avenue for regulating leukaemic cell homing and migration. IntroductionAcute lymphoblastic leukaemia (ALL) refers to a group of malignancies arising from lymphoid progenitors that may be of B-or T-cell lineage (B-ALL or T-ALL), resulting in proliferation and expansion of lymphoid blasts in bone marrow, blood and other organs. ALL has an overall incidence of 1-1.5 per 100,000 individuals and exhibits a bimodal age distribution with an early peak between ages 2-5 years (4-5 per 100,000 individuals, accounting for 80% of all childhood leukaemia), followed by a second peak after the age of 50 years (1). The treatment of ALL involves complex therapeutic programmes using intensive combination chemotherapy in dose-and timespecific sequences resulting in survival rates greater than 80% in children (2). However, there is a need for novel treatment options as only 30-40% of adults achieve long-term diseasefree survival and both childhood T-ALL and relapsed ALL are associated with poor clinical outcome (3,4).Microtubule targeting agents (MTAs) have been shown to be effective against ALL cells and vincristine is a key component of induction, consolidation and maintenance treatment protocols (3). We have previously described a novel MTA, pyrrolo-1,5-benzoxazepine-15 (PBOX-15), which exhibits anticancer activity against a variety of human tumour cell types, including those derived from both solid and haematological malignancies (5-10). Importantly, PBOX-15 and other related PBOX compounds display minimal toxicity against normal human blood and bone marrow cells, and are well tolerated by both tumour-bearing and healthy mice (7,11).MTA's interference with tubulin dynamics during mitotic spindle formation is well established; however, due to the critical role of microtubules in cell motility and homeostasis, the activity of MTAs may extend beyond inhibition of cytokinesis and subsequent induction of apoptosis. For example, we have...

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Section: Discussionmentioning

confidence: 79%

The microtubule targeting agent PBOX-15 inhibits integrin-mediated cell adhesion and induces apoptosis in acute lymphoblastic leukaemia cells

Lysaght

Verma

Maginn

et al. 2012

International Journal of Oncology

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“…An increase in tubulin detyrosination during epithelialto-mesenchymal transition (EMT) in immortalized human mammary epithelial cells promotes morphological alterations, which results in enhanced reattachment to endothelial cells (Whipple et al, 2010). However, excess tubulin detyrosination in mammalian TTL-null fibroblasts results in defects in both spindle positioning during mitosis and cell polarization during interphase (Peris et al, 2006).…”

Section: Discussionmentioning

confidence: 99%

Tubulin detyrosination promotes monolayer formation and apical trafficking in epithelial cells

Zink

Große

Freikamp

et al. 2012

Journal of Cell Science

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SummaryThe role of post-translational tubulin modifications in the development and maintenance of a polarized epithelium is not well understood. We studied the balance between detyrosinated (detyr-) and tyrosinated (tyr-) tubulin in the formation of MDCK cell monolayers. Increased quantities of detyrosinated microtubules were detected during assembly into confluent cell sheets. These tubules were composed of alternating stretches of detyr-and tyr-tubulin. Constant induction of tubulin tyrosination, which decreased the levels of detyr-tubulin by overexpression of tubulin tyrosine ligase (TTL), disrupted monolayer establishment. Detyr-tubulin-depleted cells assembled into isolated islands and developed a prematurely polarized architecture. Thus, tubulin detyrosination is required for the morphological differentiation from non-polarized cells into an epithelial monolayer. Moreover, membrane trafficking, in particular to the apical domain, was slowed down in TTL-overexpressing cells. This effect could be reversed by TTL knockdown, which suggests that detyr-tubulin-enriched microtubules serve as cytoskeletal tracks to guide membrane cargo in polarized MDCK cells.

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“…The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance [9][10][11][12] . The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells.…”

Section: Introductionmentioning

confidence: 99%

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Rahim

Üren

2011

JoVE

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Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host [1][2][3] . Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors 4,5 . The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phasecontrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process 6 . Here, we describe an electricalimpedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells. Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes 7,8 . The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance [9][10][11][12] . The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods. Video LinkThe video component of this article can be found at https://www.jove.com/video/2792/ Protocol 1. Preparation 1. All steps should be performed under sterile conditions in a tissue culture hood. 2. The xCELLigence station is placed in a 37°C incubator in the presence of 5% CO 2 . 3. Use low passage HUVEC cells, preferably no more than passage 6. Also, make sure tha...

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Epithelial-to-Mesenchymal Transition Promotes Tubulin Detyrosination and Microtentacles that Enhance Endothelial Engagement

Cited by 127 publications

References 47 publications

The microtubule targeting agent PBOX-15 inhibits integrin-mediated cell adhesion and induces apoptosis in acute lymphoblastic leukaemia cells

The microtubule targeting agent PBOX-15 inhibits integrin-mediated cell adhesion and induces apoptosis in acute lymphoblastic leukaemia cells

Tubulin detyrosination promotes monolayer formation and apical trafficking in epithelial cells

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

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