Critical to modelling the sequence of events of the cell cycle process is the ability to undertake high spatio-temporal resolution monitoring of the cell cycle progression using green fluorescent protein (GFP) cell cycle phase markers (CCPMs). This monitoring technique allows the continuous tracking of single cell checkpoint transitions within a heterogeneous population in a non-destructive (non-invasive) manner. The GFP-based probe has expression, localisation and destruction characteristics that shadow the dynamic behaviour of the Cyclin B1 regulator (during the cell cycle) in living cells [159]. That is, the status of the cell cycle is monitored and reported by dynamic stealth sensors based on the expression, localisation and degradation of GFP under the control of one of the key components of the cell cycle regulators, the Cyclin B1 protein. These sensors can precisely and non-invasively determine the status of the cell cycle in living cells by fluorescence imaging, i.e., without perturbing the progression of the cell cycle. The performance of the non-invasive stealth reporter has been validated on high content to high throughput detection platforms including multi-well high-throughput screen (HTS) imaging, single cell kinetic-tracking and multi-parameter flow cytometry [107, 159, 208]. HTS is a method of scientific experimentation used particularly in the field of drug discovery and the related fields of biology and chemistry. This method allows researchers to rapidly conduct millions of biochemical and pharmacological tests.
Cyclin B1-GFP tracking provides important sub-phase information about the progression of the cell cycle, the dynamics of the cell-cycle regulator (Cyclin B1) in parallel with morphological landmarks and DNA content analysis (Figure 5.1). Moreover, this time- lapse microscopy provides a means for understanding the subtleties in cell cycle event patterns such as delayed versus arrested patterns [209, 210]. The continuous progression of the cell cycle traverse and encoded molecular readouts in bifurcation cell lineages have been tracked to enable data extraction with subsequent linking to phenotypic cellular behaviour in both normal conditions (i.e., in the absence of TPT), and in response to treatment with the anti-cancer drug TPT (Chapter 6). In experimental terms, a cell lineage is defined as a descent in a line from a mutual progenitor (origin or starting cell) that was exposed to a given influence for a discrete time period [211]. The behaviour of both the
progenitor and the descending line of offspring (descendants) demonstrates the time- integrated cell cycle response (in the absence of the anti-cancer drug TPT) and dynamic response (in the presence of TPT) [210, 212]. Examples of such responses include changes in the inter-mitotic time (IMT), i.e., the temporal distance between two consecutive cell divisions, as well as cell death [211]. The experimental data provide the essential data upon which the cell cycle model is both developed and validated to a certain degree.
(a) (b)
Figure 5.1: Human osteosarcoma cell (U-2 OS cells): (a) Expressing a Cyclin B1-GFP stealth reporter. (b) A corresponding transmission image to identify all the cells in the field of view (taken from Reference [107]).
The parental cell line used in this thesis was a human osteosarcoma cell line (U-2 OS) derived from a 15 year old Caucasian female. U-2 OS cells were transfected (by the project collaborators at Cardiff University) with G2M CCPM (GE Healthcare, UK) using the
transfection reagent Fugene (Roche). Tracking cell cycle progression using the high- resolution fluorescence microscopy was performed with cells seeded into a multi-well coverslip-bottomed plate. Each well represents a different treatment regimen (a dose range for the anti-cancer agent TPT of 1-10 µM, see Section 6.2) in addition to a control well (untreated cells). Cyclin B1-GFP data used for estimating the unknown parameters of the extended cell cycle model (see Sections 5.3, 5.4 and 5.5) are for untreated single human osteosarcoma cells. The cultured dishes were placed onto a time-lapse instrument designed to capture bright-field phase images and Cyclin B1-GFP fluorescence images. Sequences of images were captured every 20 minutes for 48 hours (h), in triplicate, i.e. three regions of interest (ROI’s), in the control experiment.
For the cell of interest, in each image frame, three ROIs are used to extract parameters from the raw image sequence (using FLUROTRAK, a bespoke software tool by the Cardiff
collaborators) viewed in the image analysis software METAMORPH. The first ROI is
positioned on the nucleus and the other two ROIs are positioned on the cytoplasmic regions (usually, on the opposite sides of the nucleus of the cell of interest). Once the ROIs are placed, the cell of interest is tracked frame by frame for 48 h (resulting in a total of 144 frames) starting at frame one. A normal cell division results in two daughter cells, however in some cases, a progenitor cell can abnormally generate three or four individual daughter cells, i.e., cells can produce re-fused and/or polyploidy (when cells have more than two pairs of chromosomes) outcomes. The extended cell cycle model in this thesis looks into cells that experience normal cytokinesis (see Section 3.3) and such outcomes (i.e. re-fused or polyploidy) are not in the scope of this work.
0 5 10 15 20 25 30 35 40 45 50 0 100 200 300 400 500 600 700 800 Time (hours) C yc lin B 1- G F P In te n si ty (a .u .) Nucleus Cytoplasm region 1 Cytoplasm region 2
Figure 5.2: Continuous Cyclin B1-GFP intensity profile track extracted from an untreated G2cell, three compartments were tracked, the nucleus (blue) and two regions of interest in
the cytoplasm (red and green) respectively.
Continuous cell cycle tracking at the single cell level (i.e., a typical time-lapse microscopy image sequence) shows cells traversing the cell cycle and fluorescence changes (see Figure 5.2) as the cell progresses to M phase from G1. Individual cells rapidly increase Cyclin B1-
GFP expression (i.e. become brighter) and a translocation event from the cytoplasm to the nucleus compartment occurs (prior to mitosis) via the cytoplasmic retention signal [213]. That is, the fluorescent G2M CCPM reporter system relies on the control of location and
levels of expression of GFP (driven by the promoter region and removal via the destruction box) as cells progress to the later phases of the cell cycle and negotiate entry and exit to the M phase. This is attained by utilising the functional components from the Cyclin B1 protein to confer switch-like properties to the stealth reporter. Expression levels of Cyclin B1 are tightly regulated and act as a control switch that is appropriate for transition from S phase through G2into mitosis (and cytokinesis). Due to the absence of the cyclin box from
the reporter, the stealth sensor does not interfere or perturb the progression of the cell cycle.
A typical cell lineage over 48 h illustrates cellular information at two levels: (a) phenotypic behaviour (cell division represented by M) and (b) Cyclin B1-GFP reporter readout, i.e., fluorescence intensities in arbitrary units (a.u.) and hence, position in the cell cycle process at the single cell level as shown in Figure 5.2. The continuous progression of the cell cycle traverse between the two landmarks, represented by the mitotic events (M1and M2) at both
ends was demonstrated for a typical cell in the G2phase of the cell cycle.