Prevention of entrance into G2 cell cycle phase by mimosine decreases locomotion of cells from the tumor cell line SW480
Introduction
Malignant tumor cells are characterized by uncontrolled cellular growth and they gain the progressive ability to migrate from the primary site to distant organ systems. It had always been presumed that proliferation and migration do not occur simultaneously because actin filament polymerization and dynamics play important roles in both, mitosis as well as migration. In proliferating cells, the mitotic spindle assembles at metaphase and disassembles after chromosome segregation [1]. The actual cell division depends on precise actin polymerization and regulation of assembly and disassembly of the actin cytoskeleton [2], [3]. In migrating cells vectorial assembly of actin subunits into linear polymers at the leading edge and the crosslinking of filaments by bifunctional proteins is required. The disassembly of the crosslinked filaments into short fragments or monomeric subunits away from the leading edge is also necessary for cellular movement [4], [5].
In all active phases of the cell cycle (G1, S, G2, and mitosis) the human nuclear antigen defined by monoclonal antibody Ki-67 can be detected, but it is absent from resting cells (G0) [6]. As the expression of the Ki-67 protein is strictly associated with cell proliferation it is widely used as a proliferation biomarker. The cell staining intensity is low in G1 and increases to peak levels in cells in the G2 and M phases of the cell cycle [7], [8]. Measurement of the proliferative activity of tumor cells from biopsies using Ki-67 has become a well-established tool in diagnosis and prognosis during the last decade [9], [10]. Identifying the acute cell cycle phase using Ki-67 has not frequently been employed in cells which perform other cellular activities like, e.g. migration.
Multiple genetic changes are a prerequisite for the conversion of normal cells into cancer cells. These changes involve the p53 protein, which is mutated in a wide variety of human cancers [11], also in the colon adenocarcinoma cell line SW480 used in this study [12]. In normal cells p53 is induced by DNA damage and plays a role in the G1-S checkpoint pathway [13] and in triggering apoptosis [14]. Cell cycle checkpoints play a major role in the maintenance of genetic stability [15]. Therefore, the components involved in genome transmission during cellular replication and segregation (DNA, spindle, spindle pole) are under surveillance of these control points. As the defect in proliferation control is a characteristic of tumor cells, chemotherapy is mainly based on disruption of spindle formation or DNA replication. A number of substances are known to arrest cells in defined phases of the cell cycle. The plant amino acid mimosine was found to reversibly block an actively dividing asynchronous cell culture not only at the G1-S checkpoint but also in early and middle S phase. Mimosine appears to be very effective at preventing initiation of replication forks when it is delivered during the G1 period whereas it inhibits subsequent DNA replication when added in early and middle S phase [16]. These observations support the conclusion that mimosine prevents cells from entering G2 phase.
It has previously been shown that cell proliferation and migration can be influenced by the same substances e.g. growth factors [17], chemotherapeutic agents [18], or the interaction with extracellular matrix [19]. This observation indicates that the two processes are at least partly connected by signaling pathways or they are interacting with each other.
Migration is a dynamic process which is characterized by direction, time, and distance of cell movement. In the present study, we used continuous single cell analysis [20] to observe migrating SW480 tumor cells for a time period spanning more than a whole cell cycle to assess possible effects of cell cycle phases on migration. Furthermore, we investigated whether arresting tumor cells in the cell cycle pharmacologically (using mimosine) influences migratory activity.
Section snippets
Cell line and culture conditions
The cell line SW480 (human colon adenocarcinoma) was obtained from American Type Culture Collection (ATCC, Rockville, MD). The cells were maintained in Leibovitz's L-15 medium supplemented with 10% fetal calf serum (fcs) at 37°C in humidified atmospheric air without CO2 addition.
Cell migration assay
Cell migration studies within a three-dimensional collagen matrix were performed as previously described by Friedl et al. [20] with modifications. Briefly, 5.0×104 cells were suspended in 100 μl liquid bovine dermal
Results
Locomotion of cells from the human colon adenocarcinoma cell line SW480 was investigated by time-lapse videomicroscopy in a three-dimensional collagen matrix. Video images from a 72 h recording are shown in Fig. 1. This interval includes two cell divisions of one cell and corresponds to more than one full cell cycle. The selected cell, characterized by an elongated shape at the beginning (0 h), moved and started rounding up (9 h) and finally stopped migration after another hour. The parental
Discussion
Cell migration and proliferation are two characteristics of tumor cells. Both cellular activities can be directly visualized using time-lapse video recording of cells within a three-dimensional collagen matrix. The migratory activity of individual cells can be documented by this method which has already been done using a number of different cancer cell lines [22], [23], [24]. Cells from the colon adenocarcinoma cell line SW480 were also found to migrate spontaneously [25]. Migrating SW480 cells
Acknowledgements
The authors wish to thank J.C. Feldner for help. We are grateful to PD Dr C. Lang for useful discussions and for critical reading of the manuscript. This work was supported by the Fritz-Bender Foundation, Munich, Germany and Westdeutsche Landesbank Girozentrale Düsseldorf, Germany.
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