Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.)

Cell morphological changes combined with biochemical assays for assessment of apoptosis and apoptosis reversal. Chaitanya Joshi1, Bharat Karumuri1, Jamie J. Newman1 and Mark A. DeCoster1,2 1

Biomedical Engineering and 2Institute for Micromanufacturing, Louisiana Tech University, Ruston, Louisiana, USA 71270

It has recently been reported that apoptosis induced in cancer cells can be reversed if a sub-maximal apoptotic stimulus is removed. To investigate the effect of apoptosis reversal on cell morphological changes, we induced apoptosis in brain tumor cells as well as normal glial cells, specifically astrocytes, using the apoptosis inducer staurosporine (STS). Regular apoptotic changes and cell death were noted in both brain cell types treated with high concentrations of STS. However, we observed apoptosis reversal with regrowth of some cancer cells with sub-maximal STS treatment. We also observed that morphology of the cells changed after the stimulus was removed and apoptosis was reversed, as demonstrated by continued cell proliferation and viability staining. Early apoptotic changes in cell shape and morphology were monitored using calcein, a viability stain, and live cell fluorescence microscopy. Nuclei were identified using DAPI staining to accurately determine changes in the nuclear area factor (NAF), a factor we have previously described as an excellent indicator of early apoptosis. Captured microscopy images were analyzed using the image analysis program Image Pro Plus. The biochemical (MTT) assay was also used as a measure of metabolic activity for larger cell populations during apoptosis. By combining both imaging and biochemical assays, we have demonstrated a clear reversal of apoptosis in brain tumor cells as indicated both by cell morphological and metabolic changes, respectively. The limited ability of normal brain astroctyes to respond to apoptosis reversal conditions compared to cancer cells is also demonstrated. Key words: Image analysis; astrocytes; glioma; brain tumor; apoptosis reversal

1. Introduction Cancer cells display a number of unique characteristics that allow them to evade normal biological processes and thus to compete with neighboring cells for resources and survival. These characteristics include cancer cells’ ability to continuously proliferate without undergoing senescence and ability to continue dividing despite cellular damage. The decrease in programmed cell death seen in cancer cells makes them harder to treat with standard chemicals/drugs that damage cells and in turn lead to apoptosis. Instead, these cells are largely resistant and so require large amounts of cytotoxic chemicals to destroy them. Our studies indicate that not only do cancer cells require a high concentration of these chemicals, but the cells also require long exposure in order to confirm complete cell death. Apoptosis, or programmed cell death, occurs both in normal developmental processes [1] as well as in disease [2]. Recent studies have demonstrated that cancer cells can be resistant to apoptotic stimuli and might also have the ability to survive the early stages of apoptosis and return to their viable cell state if the stimuli are removed [3, 4]. Here we use imaging and metabolic assays to demonstrate that the phenomenon of apoptotic reversal is a favored property of cancer cells, at least in comparison to the normal brain astrocytes used here as controls. Imaging assays were used to determine changes in cell viability, cell shape, and nuclear shape during and after exposure to apoptotic stimuli. Apoptosis causes visible changes in all of these cellular phenotypes and so high-quality imaging can be a powerful tool to track the progression of apoptosis. In addition, we know that cancer cells proliferate rapidly and so also have a high metabolic rate, and that as cells undergo apoptosis, metabolism decreases as a result of slowed proliferation and the beginning of programmed cell death. Therefore, we can use these changes in metabolism as another metric to monitor the apoptotic progression of these cells over time, which we did here using a biochemical assay. Our data demonstrate that there is a window during exposure to apoptotic stimuli in which the appropriate cell number and drug concentration can lead to apoptosis if presented to the cells for an extended period of time. However, if the stimulus is removed early, the cells are able to recover from this apoptotic state and continue to grow and proliferate. This phenomenon of apoptotic reversal is demonstrated both visually and quantitatively. We provide images of cell morphology over time, which clearly demonstrate apoptotic changes that are reversed when the stimulus is removed from the cells. We also provide quantitative measurements of metabolic rate, demonstrating an increase in metabolic rate for cells that have survived the initial exposure to apoptotic stimuli. Also, in a direct manner using live cell imaging with an automated incubator microscope system, we observed recovery and proliferation of brain tumor cells following removal of apoptotic stimuli for the same field of cells over time.

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2. Materials & Methods 2.1 Cell Culture Rat brain glioma cells (CRL 2303) were obtained from and cultured according to vendor specifications (American Type Culture Collection, Manassas, VA) in DMEM medium supplemented with 10% fetal bovine serum and penicillin/streptomycin. Rat brain astrocytes were isolated from newborn rats and grown in Kaighn’s F-12 Ham modified basal media supplemented with 5% each of fetal bovine and horse serum, and penicillin/streptomycin as we have previously described [5]. All cells were maintained at 37° C in a humidified incubator containing 5% CO2 and retired by passage 20. Cells were plated in standard 24-well plates and incubated for 20-24 hours before subjecting to apoptotic stimulus. For experiments tracking morphological changes cells were plated at densities of 15,000 cells/ml or 7,500/cm2. For biochemical assays (MTT), cells were plated at densities of 40,000 cells/ml or 20,000/cm2. Apoptotic stimulus was applied using staurosporine (STS) as described previously [6] at 2 and 0.5 µM. Dimethyl sulfoxide (DMSO) in Locke’s solution was used as a negative vehicle control for all experiments. The apoptotic stimulus was removed from certain wells after 8 hours and was replenished with fresh warm growth medium to track the reversal phenomenon. 2.2 Cell staining and imaging Preliminary experiments were carried out using light microscopy. Images were captured using a live cell imaging system (IncucyteTM, Essen Bioscience) at different time points before STS treatment, during STS treatment, and after washing STS off the cells. Calcein AM (Invitrogen) was used to carry out fluorescent staining in living cells to establish cell area factor (CAF) which has been previously described as a marker of early stages of apoptosis [6]. A primary loading solution for calcein was freshly prepared using 85 µl of pre-warmed Locke’s solution incubated at 37°C and 5 µl of pluronic acid in DMSO (20% weight/volume). Ten µl of Calcein stock with a concentration of 1 mM was added to the above solution to prepare 100 µM primary loading solution. Before staining, the growth medium was aspirated from each well and replaced with 495 µL of pre-warmed Locke’s solution. Five µl of calcein loading solution was added to each well and then cells were incubated for 10-15 minutes. Fluorescence microscopy was carried out using an Olympus inverted epifluorescence IX-51 microscope using a 485 nm excitation filter and a color camera. Images were captured at 80-125 msec camera exposure to get the best signal to noise ratio images. 4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI) was used for identifying cell nuclei as previously described [7]. Loading solution for DAPI was prepared from 14.27 mM stock solution by adding 10 µL of stock solution to 990 µL of Locke’s solution. Growth medium was aspirated from each well and replaced by 200 µL of Locke’s and 50 µL of DAPI loading solution. The cells were then incubated for 30-60 minutes before imaging was carried out on an Olympus inverted epifluorescence IX-51 microscope with color camera. Image enhancement tools like Black Balance, and color level adjustments were used during imaging to improve image quality. The cells were alive in both of the staining methods, which allowed imaging with natural morphology of cells, eliminating any morphological changes that would be due to fixation. 2.3 Biochemical Determination of Apoptosis using Mitochondrial/MTT assay Quantitative analysis of cell metabolism was done using a colorimetric assay with MTT [3-(4, 5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide], using modifications from our previously described method [8]. Readings were taken at 5 time points: the first was taken before treatment to establish a basal level of metabolic activity, the second and third were taken at 3.5 and 7 hours, respectively. The final two readings were taken after 3 and 11 hours post washing off of the apoptotic stimulus. MTT stock solution was made (1.25 mg/ml in Locke’s Solution) and was pre-warmed before adding to the cells. Media was removed from the well and 400 µL of MTT stock solution was added. Cells were incubated for 75 minutes at 37°C. MTT solution was removed after 75 minutes, and 250 μl of 91% isopropanol was added to the wells. The non-water soluble violet formazan crystals dissolved into the isopropyl alcohol, and the amount of these crystals was determined spectrophotometrically which served as a quantitative estimate for cellular metabolism. Two hundred μl of isopropanol with dissolved formazan crystals was transferred into a 96 well plate and the optical density (OD) was measured at 570 nm wavelength with a spectrophotometer (Thermo Scientific Multiskan ® Spectrum). 2.4 Image processing using Image Pro Plus Image Pro Plus V6.1 was used to analyze Calcein and DAPI staining images. The object counting tool was used to count cells which recognized bright objects against a dark background. Object area, count, and roundness were the selected parameters which were later used for calculation of Cell Area Factor (CAF) and Nuclear Area Factor (NAF) for Calcein and DAPI staining images, respectively as previously determined [6, 7, 9]. Since glial cells and cancerous glia tend to form network in cultures, it was neccessary to differentiate single cells as individual objects from a clump of

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cells or connected cells. ‘Split objects‘ function was used to distinguish cells touching each other. This function was used only for Calcein stained images since the cells are entirely stained, whereas DAPI specifically stains the nucleus and thus was unlikely to show objects touching each other. The resultant object areas were obtained in (pixel)2 units and roundenss values were unitless. The average area/object was calculated by summing up all areas and dividing by the number of objects per image and average object roundness was calculated by suming up all roundness values and dividing by the number of objects. CAF was calculated by multiplying the average area/object obtained from calcein stained images and average object roundness. NAF was calculated by multiplying the average area per object obtained from DAPI stained images and average object roundness. The roundness measurement in Image Pro Plus is calculated using the formula: (perimeter2)/(4*pi*area), with 1.0 indicating a perfect circle and larger values indicating oblong and non-circular objects. As the cells undergo apoptosis, the overall cell and its nucleus shrink and become more round which leads to a decrease in average area of the cell and nucleus and so the roundness value will tend to move closer to 1.0. Thus as apoptosis proceeds, both CAF and NAF will become smaller in value, and then larger again as recovery occurs.

3. Results & Conclusions 3.1 Morphological and Biochemical Changes During Apoptosis As we have previously described, cell morphological changes can be an early indicator of responses to apoptotic stimuli [6, 7, 9]. While these changes may be easily seen with white light and phase microscopy, returning to the same image field can be a challenge and extended imaging between time points by removing cells from the incubator certainly adds stress to the cells, making the response less relevant than would be expected in vivo. Balancing this, new instrumentation including live cell imaging systems that retain incubator conditions is now available [10]. We used live cell imaging to trace morphological changes in rat glioma cells over time for normally growing cells, cells induced to undergo apoptosis with STS, and cells treated with STS and then washed with growth media to investigate reversal of apoptosis (Figure 1).

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Figure 1. Recorded morphological changes over time in glioma cells. Rat glioma cells were treated with a vehicle control, treated with 500nM STS, or treated with 2uM STS. The top panels show cells that were treated for a total of 19 hours. The bottom panels show cells that were washed 8 hours after treatment and show clear morphological reversal of apoptosis even 3 hours after stimulus is removed (11 hour time point). Scale bar indicates 100 microns.

Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.)

Control cells show a robust growth as indicated by the cell culture surface filling in over time from 0-19 hours (top rows, Fig. 1). Both 500 nM and 2 µM concentrations of STS cause rapid cell morphological changes, with the higher concentration (2 µM) resulting in a more complete rounding of cells, suggestive of further progression towards apoptosis. At both concentrations, few cells are being lost from the field of view, so cells are remaining attached to the culture surface during these apoptotic changes (middle and bottom rows of Fig. 1). However, a striking difference that is seen between 500 nM and 2 µM STS is the reversal of apoptosis that results when STS is removed 8 hours posttreatment. As shown in the bottom set of panels in Figure 1, washing with growth media at 8 hours post treatment results in continued growth of control cells (top row, bottom set of panels). There is some morphological recovery of cell shape for cells treated with 2 µM STS, but a much greater spreading of cell area for cells treated with only 500 nM STS, an indication of recovery after washing out the lower STS stimulus (Fig. 1, middle row of bottom set of panels). In considering these morphological changes, we next determined whether a biochemical assay could indicate reversal of apoptosis induced by STS. As shown in Figure 2, using the MTT metabolic assay, we measured a robust increase in cell growth in the glioma cells of over 300% in control samples by 19.5 hours (Fig. 2A).

Figure 2. Metabolic assay demonstrates increased activity corresponding with apoptotic reversal. MTT assays were performed on both rat glioma and rat astrocyte cells in the presence of STS. As described in Figure 1, cells were either left exposed to STS for 19 hours or the stimulus was removed after 8 hours, and then cells were allowed to continue growing in fresh media. Note difference in Y-axis scale for Glioma (A) and Astrocyte (B) data, indicating much higher growth for glioma cells. Data is represented as percentages relative to an untreated control, and data represent averages with standard deviation for each treatment shown.

In general, glioma cells treated with STS displayed decreasing MTT values over time. However, by 19.5 hours posttreatment, cells treated with 500 nM STS were showing some spontaneous recovery, a feature not observed in cells treated with 2 µM STS. This recovery from apoptosis was greatly enhanced when the apoptotic stimulus was removed and cells were given fresh media (bars right of dotted line). While the 11 hour timepoint (3 hours post-wash) did not show perceptible differences over its time-matched condition with no wash, at 19.5 hours (11.5 hours post-wash), the lowest STS treatment of 500 nM demonstrated much higher MTT values than its unwashed 19.5 hour treated value. As expected, the higher concentration, 2 µM STS, condition showed less recovery than did 500 nM. This data further supports the need not only for higher doses of apoptotic drugs in cancer treatments, but also the need for extended windows of treatment. Since the abnormal proliferation of brain glia including astrocytes is thought to play a role in the transition to (glioma) brain tumors [11], we investigated the potential for astrocyte recovery from apoptotic stimuli (Fig. 2B). As shown by the MTT values, astrocytes as expected demonstrate a much lower metabolic growth rate than do glioma cells, with approximately 150% growth after 11 hours of plating. In fact at 20 hours post plating the MTT assay indicates slightly less metabolic activity, possibly indicating a more quiescent state for the astrocytes. After STS treatments, for both 500 nM and 2 µM, MTT values continue to decrease over time compared to their matched time controls treated with media only. It is interesting to note that for both the 11 hour and 20 hour post-wash conditions that astrocytes washed only with media (and not receiving STS) showed a boost in MTT values, which was lacking in the glioma cells. This may indicate that without fresh growth factor addition, normal brain astrocytes may more quickly enter a phase of shallow growth or quiescence compared to highly proliferative glioma cells. It is also interesting to note that after washing out the STS apoptotic stimulus, astrocytes showed recovery in MTT levels at both early (3 hours post) and late (12 hours post) time points. However, in contrast to glioma cells, the recovery for astrocytes was less at the later time point, indicating a possible shortened and limited recovery potential for astrocytes as compared to glioma cells. To our knowledge, this is the first time this comparison has been noted, and may indicate the increased ability of glioma cells in brain tumors to sustain recovery from damage compared to normal brain glia. 3.2 Determination of Cell Area Factor as a Measure of Apoptosis Calcein AM is a vital fluorescent dye, which has been used extensively with microscopy [6, 12, 13] to determine cell viability. The fluorescent moiety is conjugated to an acetyl methyl ester, which aids in loading the living cells and

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blocks the fluorescence until the ester is cleaved by naturally occurring esterases inside the cell. In this manner the cleaved dye is also trapped within the cell after esterase action. Once cells are loaded with the dye, living cells fluoresce brightly. With little or no fluorescence outside the cells, images can be generated with very good resolution, and little or no background. Fluorescence microscopy is known for its potentially high signal-to-noise ratio, and calcein staining is a powerful stain, as some other fluorophores must be washed out of the sample due to the fact that they are intrinsically fluorescent. Calcein AM loading allows for specific fluorescent labeling within living cells, and we have previously used this probe to track dynamic cell morphology changes during apoptosis [6] and to derive a measurement of a quantitative morphological change we call the Cell Area Factor (CAF). Here we utilized CAF to measure reversal of apoptosis following removal of the STS stimulus. As shown in Figure 3, reminiscent of the phase images shown in Figure 1, removal of the STS pro-apoptotic stimulus shows a greater recovery of cell surface and less rounded cell morphology for the lower STS concentration compared to the higher concentration. Because of the greater signal-to-noise potential and ease of image analysis assisted identification of discrete objects, the calcein images are much more straightforward for deriving CAF than would be the case for white-light microscopy images. Quantitative measurements of CAF are shown in Figure 3 (C+D), demonstrating dramatic reversal of apoptosis as indicated by increased CAF values for glioma cells after removal of STS. As expected, CAF values drop more steeply with time when cells are treated with 2 µM STS as compared to 500 nM. In addition, determination of CAF demonstrates a much more dramatic apoptotic reversal following the removal of STS in cells treated with the lower concentration of stimulus (Fig. 3C), a result similar to what was observed using the MTT assay (Fig. 2A). These data suggest that under these conditions, CAF is not only an excellent marker for early morphological changes, but also a metric for identifying apoptosis reversal and recovery. A comparison of the data between the CAF determination (Fig. 3) and the MTT results (Fig. 2) suggests that CAF indicates recovery and apoptosis reversal sooner than MTT. This may indicate an “outside-in” sequence of events whereby removal of an apoptotic stimulus enables morphological changes before the inside biochemical changes can begin to recover.

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Figure 3. Quantification of morphological changes in glioma cells using imaging and determination of cell area factor. A+B: Images were taken at different points before and after addition of STS. Seven hours after the initial stimulus cells were either allowed to continue growing in that stimulus until 18 hours or cells were washed and provided fresh media in the absence of STS and allowed to grow an additional 18 hours (18 hrs. + wash). Scale bar indicates 100 microns. Row A= glioma cells; Row B= astrocytes. C+D: Graphs depict the data for measured CAF over time for both rat glioma cells (C) and normal rat astrocytes (D). CAF on the Y-axis depicts units in pixels2, with averages and standard deviation shown for each treatment. © 2012 FORMATEX

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Compared to glioma cells (Fig. 3A+C), rat brain astrocytes showed much larger cell bodies as indicated by CAF (Fig. 3B+D). On average, the CAF for control astrocytes (Fig. 3D) was at least 4 times greater than the CAF for glioma cells (Fig. 3C). A very large percentage of this CAF was lost after treatment of the astrocytes with STS, and in contrast to the glioma cells (Fig. 3C), only about 50% of the CAF on average was regained by astrocytes after removal of STS by 11 hours post-wash (19 hour time point, Fig. 3D). The results from astrocytes further confirm the unique capabilities of cancer cells to more quickly and effectively recover from apoptotic stimuli, demonstrating the challenges for optimisation of treatment conditions to effectively treat cancer. 3.3 Determination of Nuclear Area Factor as a Measure of Apoptosis Apoptosis is known to induce nuclear changes in the cell [14, 15], and we have previously shown that the nuclear area factor (NAF) is an excellent marker for early apoptotic changes including those associated with DNA fragmentation [7, 9]. Here we investigated whether reversal of apoptosis by removal of STS stimulus could be observed by monitoring changes in the NAF. As shown in Figure 4, NAF decreases continuously with time for the higher (2 µM) STS treatment, but is somewhat delayed for the lower (500 nM) treatment.

Figure 4. Quantification of nuclear morphological changes (nuclear area factor, NAF) in glioma cells and brain astrocytes during apoptosis and apoptosis reversal. Graphs depict the data for measured NAF over time for glioma cells (A) and brain astrocytes (B) treated with indicated concentrations of staurosporine (STS) and recovery effect (reversal of apoptosis) after post-wash removal of STS. Averages and standard deviations are shown for each treatment.

Furthermore, it appears that despite the continued presence of STS at the later time points, the progressive decrease in NAF in glioma cells diminishes, an indication that cells may be trying to survive. As expected, the reversal trend for NAF was higher for cells treated with 500 nM STS than for those treated with 2 µM STS, and indeed, by the 19 hour time point the average NAF value for cells treated with 500 nM STS and recovered, was almost exactly at control (time 0) values (Fig. 4A). Similarly to the CAF, the NAF for astrocytes (Fig. 4B) was larger than for glioma cells (Fig. 4A). Furthermore, NAF values continued to drop at all time points measured for astrocytes (Fig. 4B) and showed modest reversal compared to glioma cells (Fig. 4A). Reversal as indicated by NAF was estimated to be at most 60% by 19 hours for astroctyes (Fig. 4B), while it was close to 90% for glioma cells (Fig. 4A). Together, these data demonstrate that we can use various metrics for determining apoptotic progression and reversal in both cancer cells and normal cells, all pointing to a more dramatic reversal of apoptosis in cancer cells when compared to normal cells.

4. Discussion The ability for rat brain glioma cells to display more dramatic recovery from apoptosis when compared to rat brain astrocytes, as we have shown here, may be one mechanism by which tumor cells out-compete their healthy neighbouring cells in the brain. Furthermore, it has been shown that brain tumor cells release glutamate and use it as an autocrine factor [16], while simultaneously damaging normal brain cells such as neurons, further gaining tumor cells a competitive advantage in the brain. In considering these dynamics in the brain and our work here, a combination of glutamate autocrine function and apoptosis reversal potential may provide brain tumor cells growth advantages in the brain. Figure 5 provides an overall view of considerations at the cellular level for apoptosis progression and the potential for reversal in cancer including cell density, apoptotic stimuli concentration, and time. From left to right in the figure, with increasing time and concentration of the apoptotic stimulus, initial apoptosis initiation and reversal checkpoints move from early apoptosis to late apoptosis, which may then become irreversible. Our studies suggest an optimal balance for these factors where exposure time and drug concentration determine the ability of cells to recover. In addition, higher cell densities dilute the apoptotic effect in cell culture and may also provide protection for brain tumor cells by increasing the local concentration of protective autocrine factors such as glutamate [16]. When using assays to evaluate the apoptotic cascades in cell culture, since biochemical assays often require much greater cell numbers than

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are needed for microscopy analysis, this effect of cell density on apoptotic outcome should also be considered as we suggest in Figure 5. Figure 5. Apoptosis progression and potential for reversal in cancer. Our studies establish that reversal of apoptosis is possible in cancer cells, but that this reversal is highly dependent on length of apoptotic stimuli exposure, cell density, and concentration of stimuli present. Together these 3 factors can come together at an optimal point that results in the ability of cancer cells to survive early apoptosis and return to their normal state. If the stimuli are left too long, or at too high a concentration relative to the cell density, then cells continue progressing towards apoptosis and will no longer be capable of reverting to their cancerous cell state.

Acknowledgements: The authors wish to thank Mrs. Kinsey Cotton for her helpful discussions in generation of Figure 5. We also wish to thank Dr. James Cardelli and members of his lab at LSU Health in Shreveport, Louisiana (especially David Coleman) for providing us recording time with the Incucyte videomicroscopy system. This work was funded in part by National Science Foundation grants #1032176 and #1116706, PKSFI Contract No. LEQSF (2007-12)-ENH-PKSFI-PRS-04 from the Louisiana Board of Regents, and funds from the James E. Wyche III endowed professorship provided by Louisiana Tech University (to M.D.).

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