Chemosensitivity assay in mice prostate tumor: Preliminary report of flow cytometry, DNA fragmentation, ion ratiometric methods of anti-neoplastic drug monitoring
© Sharma and Kline; licensee BioMed Central Ltd. 2004
Received: 30 December 2003
Accepted: 19 March 2004
Published: 19 March 2004
Flow cytometry, DNA fragmentation, ion ratiomateric analysis and NMR peaks characterized drug chemosensitivity of antineoplastic drugs. Hypotheses were: 1. The chemosensitive effect of different cancer cell lines is characteristic; 2. DNA fragmentation, ion ratiometric analysis suggest apoptosis status of tumor cells.
PC-3 cell lines were compared with DU-145, LNCaP cell lines in culture for the [Na]i and [Ca]i ion sensing dyes, cell death, NMR peaks and apoptosis staining for chemotherapeutic action of different drugs.
DNA fragmentation, ratiometric ions and fluorescence endlabelling plots were characteristic for cell lines and drug response. 31P-23Na NMR spectra showed characteristic high phospho-choline and sodium peaks.
Flow cytometry, DNA fragmentation, ion ratiometric methods and NMR peaks indicated apoptosis and offered in vivo drug monitoring method.
Recently, drug chemosensitivity assays were developed to evaluate anti-neoplastic drugs using cell cultures. Incorporation of cell culture studies offers good possibility as gold standard to assess the drugs due to the controlled conditions and easy procedures. Tumor cell proliferation, apoptosis, malignancy are frequently reported due to ionic and metabolite changes in tumor culture studies. These cancer cell culture techniques demonstrated apoptosis by DNA fragmentation, NMR peaks and fluorescent flow cytometry [1–5]. In the present paper we demonstrate the association of apoptosis cell death using DNA fragmentation analysis, ion ratiometric assay and NMR spectroscopy of tumor cells.
Materials and methods
PC3 human prostate cancer cells were propagated in nude mice as experimental model of prostate tumor. Cells from the dissociated tumor were put into culture. These cells were grown overnight and then subjected to radiotracer uptake, drug sensitivity, MTT analysis, flow cytometry, ratiometric ion assay and DNA fragmentation experiments as described elsewhere .
Cell survival and DNA fragmentation
Three human prostate cell lines were used in culture studies (PC-3, DU-145 and LNCaP) and tumor propagation in mice. The experiments were designed for: (a) Drugs doses – taxotere: 1, 10, 20, 50, 100 nM; 9-amino-camptothecin: 10, 20, 50, 100, 200 nM; and, VP- 16 etoposide – 1, 2, 5, 10, 20, 50, 100 μg/ml; (b) Cell lines – PC-3, DU-145, LNCaP; (c) Exposure times – 4, 8, 12 hrs; 1 and 2 days; (d) Analysis by using control wells for all drug exposure times and cell lines.
Cells were obtained from American Type Culture Collection, Rockville, Maryland, grown in RPMI 1640 media (Sigma) supplemented with 5% Fetal Bovine Serum (Sigma), and passaged every 3 days. After washing × 3 with PBS, cells were trypsinized (0.1% Trypsin and 1 mM EDTA in PBS), mixed 1:1 with serum containing medium, resuspended in PBS, resuspended in iced cold methanol free formaldehyde (1% in PBS) for 15 min, resuspended in 70% ethanol and stored at -20°C. Fixed cells were exposed to endlabelling cocktail overnight, resuspensed in "rinse buffer", exposed to fluorescent antibody and finally treatment with propidium iodide and RNAase for DNA fragmentation analysis. Flow cytometry was performed on a Becton/Dickenson FACStar II at the Columbia University Cancer Center. Individual fluorescence intensity values for 10,000 cells were obtained or each sample .
Relative sensitivity of PC3 and DU145 cell lines to VP-16 etoposide and taxotere
Relative sensitivity of PC3 and DU145 cell lines to VP-16 etoposide (10 μg/ml) and taxotere (20 nM) were examined with flow cytometry.
Fluorescent approach to the measurement of intracellular ions
Measurement of Ca++
Human prostate cancer cell line PC3 was cultured in black, low autofluorescence 96 well plates. 10 nMol/L taxotere was added to cell wells (six wells per time point) before 2, 4 and 6 hours of loading of a Ca++ sensitive fluorescent dye (Fura II/AM). The IC50 value at 24 hrs, as determined by MTT assay, was 10 nM taxotere. The ratio of fluorescent intensity for the two-excitation wavelengths (345 and 390 nm) was plotted versus duration of drug exposure. Increases in ratio imply increases in [Ca]i. The following sequence of interventions in 96 well plate were run: i. 96 wells were read at both wavelengths for dye exposed cells, unexposed cells, cell free wells and dye free/cell free wells; ii. ionophores or thapsigargin were added to dye exposed cell wells in presence of Mn++ to quench dye and obtain well by well zero values.
Measurement of Ca++ by sodium green Na+ sensing fluorescent dye
The Na+ sensing fluorescent dye was employed which PC3 human prostate cancer lines used and readily took up. Data was displayed as fluorescence intensity (vertical axis) versus forward scatter (horizontal axis) described elsewhere .
In vitro Na-NMR spectroscopy studies on superfused human PCa cells
PC-3 cells were embedded in agarose beads, which were perfused in an NMR tube. 31P and 23Na TQ spectra of perfused PC-3 cell loaded agarose beads were first acquired at baseline. Subsequently, 100-nM/L taxotere was added and 31P-23Na TQ spectra were again acquired at various times .
Cell survival and DNA fragmentation studies
Different cancer cell lines are represented for their mean % survival in presence of different drugs as shown in Figure 2A.
Drug VP 16 (Dose)
Mean % survival with VP 16(n = 5)
Drug with PC3 cells
Endlabelling Fluorescence (A.U.)
One dimensional histograms show relative number of cells (vertical axis) for fluorescent intensities over a 4 log unit range (bottom horizontal axis) using a 256 channel A/D converter (top horizontal axis). Standard errors for most samples were less than 1% of mean. Sample histograms are shown in Figure 2 for the following conditions: baseline (2A), control PC3 cells (2B), 24 hr treatment with 10 nM taxotere (2C), same with 50 nM taxotere (2D). The method had a dynamic range of approximately × 1000.
To assay DNA fragmentation (a downstream response to apoptosis), a terminal deoxynucleotidyl transferase endlabelling fluorescence approach was used. Fluorescent moieties were attached to all DNA fragment ends. Camptothecin (200 nM) treated PC-3 cells examined by APO-BRDU showed dynamic range of fluorescence intensities of up to 200 fold comparing control cell-to-cell with advanced DNA fragmentation. The combination of taxotere on PC3 was apparently saturated at 10 nM for 24 hrs at about 10% of the camptothecin value, a near maximal response (Figure 2E).
Relative sensitivity of PC3 and DU145 cell lines to VP-16 etoposide and taxotere
Relative sensitivity of PC3 and DU145 cell lines to VP-16 etoposide (10 μg/ml) and taxotere (20 nM) were examined with flow cytometry using a new fluorescent endlabelling kit. The VP-16 response was greater than taxotere for both cells lines and the DU 145 response was greater than PC3 for both drugs.
Comparison of drugs on DNA fragmentation
Dependence of DNA fragmentation is shown on drugs and cell lines using APO-BRDU endlabelling technique for various antinucleoplastic agents (VP16 etoposide, 9-amino-campothecin, cisplatin) at various times (8 and 24 hours).
Drug exposure with cell lines
APO-BRDU Endlabelling Fluorescence (n = 5)
Taxotere + DU145
Taxotere + PC3
VP 16 + DU145
VP 16 + PC3
Campothecin + DU145
Cisplatin + DU145
Dependence of DNA fragmentation time curve on cell lines
Fluorescent measurements of free [Ca++]i
Sodium green pilot studies
Sodium green dye was readily loaded in all human prostate cancer cell lines. Both ionomycin and monensin ionophores altered the sodium green fluorescence signal, and that VP-16 treated DU-145 and PC-3 cells displayed two populations on two-dimensional histograms (sodium green vs forward scatter in Figure 5B). These two populations existed after gating out necrotic cells through propidium iodide (PI) fluorescence. Two-dimensional forward scatter/orthogonal scatter histogram analysis showed that they do not occur in similar two-dimensional histograms using the Na+ insensitive, acetoxymethylester dye calcein/AM. These remaining non-necrotic populations also had different PI fluorescence.
[Na]i and [Ca]i ion sensitive dye assay
In vitro NMR spectroscopy studies on superfused human PCa cells
The current study presents the fluorescence characteristics of prostate cancer cell lines and indicative of apoptosis-rich tumor physiology in presence of anticancer drugs.
Cell survival following antineoplastics in culture
Cell survival MTT assay defined time course and dose dependence of antineoplastics on human prostate cancer cell lines. Our approach to study time points with MTT technique established the texotere dose to get IC50, IC90 (inhibitory concentration affecting 50%, 90% of cells respectively) till 24 hrs (or 48 hours if response was slow). Multiple doses and times were assessed simultaneously (e.g. 96 well plate experiments with fluorescent dyes) that support our previous study .
Quantitative measures of apoptosis with flow cytometry following antineoplastics in culture
Cell survival assays appeared to identify steps in the apoptotic pathway were based on appearance of DNA fragmentation strand breaks and total fluorescent-labeled DNA (propidium iodide) following chemotherapy. Further, this late event in apoptosis (DNA fragmentation) could be quantitated. Our Annexin V/ FITC conjugated antibody and flow cytometry observations corroborated with other study on enhanced fluorescence indicating reorientation of phosphatidylserine within the cell membrane and identification of early cellular commitment to the apoptotic cascade .
To fulfill it, experiments were designed for: (a) early events and (b) late events. Early events such as enhanced fluorescence indicated reorientation of phosphatidylserine within the cell membrane after cells were exposed to an Annexin V/ FITC conjugated antibody. One-dimensional histograms were used to examine fluorescence. Late events were evidenced as DNA strand breaks and total fluorescent-labeled DNA (propidium iodide) in fixed cells (treated and control). Two-dimensional histograms of endlabelling fluorescence and propidium iodide (cell cycle plots) provided reasonably good guide of late events and standard error on scatter in both enzyme free and secondary antibody controls using FACS data as earlier reported .
Changes in intracellular Na
Fluorescent apoptosis studies and MTT survival assays determined the effects of drugs at various duration exposures to antineoplastics in culture. The time course and magnitude of [Na]i changes appeared to indicate the events in the apoptotic cascade, and characterize the drug/dose for each cell line. SBFI/AM and sodium green dyes (Molecular Probes) appeared as best choice for fluorescent plate scanner and flow cytometry respectively . SBFI/AM (a dual excitation wavelength dye) was observed as ideal choice for cultures at low autofluorescence in 96 well plates to introduce dye, and measure average [Na]i of the cells in each well. However, this dual excitation wavelength dye was not good for standard fluorescence activated cell sorters (FACS). Sodium green for flow cytometry required standard FACS excitation and emission wavelengths. We could run suspensions of controlled and treated live cells through the FACS, to sort populations for subsequent fixation and treat them with SBFI for a confirmatory measure of [Na]i in controls or in the presence of Na ionophores.
These two approaches demonstrate reliable comparison of average [Na]i in treated versus untreated tumor cells, and confirm the apoptotic cells. Sodium green fluorescence and FACS analysis sorted out the PC3 and DU145 cells exposed to VP-16 in presence of sodium ionophore at varying [Na]o concentrations in buffer with balanced chloride and calcein/AM loaded with PI. SBFI/AM is known as dual excitation wavelength dye used with plated cells for their culture, treatment with antineoplastics and examination for the average [Na]i in each cell using the ratio method and in situ calibration. Annexin V/ FITC for fluorescence and SBFI for comparison of [Na]i in two cell populations (with or without apoptosis) interpreted mechanistic relationship between [Na]i, apoptosis and necrosis .
23Na-NMR/31P-NMR spectroscopy studies of prostate cancer cells in vitro
23Na-NMR spectroscopy of cell cultures determined the transverse and longitudinal relaxation times to assess changes in [Na]i as noninvasive technique. In our experiments, tumor cells were contained in agarose beads as reference phantom in the center of perfusion filter for suitable acquisition of TQ sodium signals and phosphorous NMR spectra for longer times and were comparable with intracellular (IC) and extracellular (EC) components of SQ and MQ signals in presence of shift reagent for chemotherapy experiments . These experiments suggested that transverse relaxation times of IC and EC compartments using evolution time dependence of the MQ NMR signal at various time points might characterize chemotherapeutic effect. 31P-NMR spectral peaks showed enhanced phosphorylcholine/choline.
In present study, dose dependence and time course using in vivo Na-NMR studies on common prostate cell lines and tumor cells standardized the rapid chemotherapeutic assay of antineoplastic drugs.
The relationship of [Na]i/[Ca]i and apoptosis in present study supported previous view on issues of Na-based data  and potentially new antineoplastics: Do [Na]i act by altering [Ca]i? Are [Na]i or [Ca]i signal changes sufficient to cause apoptosis in these cell lines? Does blocking of [Ca]i during drug exposure affect the drug efficacy? What happens to membranes potential during exposure and how is this related to [Na]i?
Fura II/AM measurements and SBFI measurements of [Na]i, were better ratiometric dual excitation dye than a flow cytometry compatible dye. Ratiometric dyes offer more accurate absolute activity reading. Our choice of ionophores for Na+ (monensin and gramicidin) and Ca++ (ionomycin) for the periods of 4 to 24 hours confirmed changes using fluorescent ratiometric dyes, fluorescent endlabelling and annexin V antibody. This approach determined the relationship between membrane changes and intracellular activity changes during apoptosis supporting recently reported literature [11–14].
[Na]i and [Ca] are represented to rise starting 2–6 hours and stay elevated for up to 24 hours following exposure of PC3 cells to the antineoplastic drugs taxotere or VP16.
Exposure Time (in hours)
Change in SBFI ratio
[Na]i change (in mM)
Normalized [Ca]i (Fura II/AM(in × 102))
1.00 ± 0.02
1.03 ± 0.02
1.02 ± 0.02
1.21 ± 0.24+
1.82 ± 0.21++
1.1 ± 0.12*
1.13 ± 0.11**
1.22 ± 0.12*
1.22 ± 0.14**
1.08 ± 0.41*
1.06 ± 0.52**
1.81 ± 0.22
1.11 ± 0.12
1.62 ± 0.21+
2.61 ± 0.32++
1.14 ± 0.21
1.52 ± 0.13+
2.41 ± 0.32++
1.09 ± 0.13
2.04 ± 0.4 1
This manuscript in part was presented at peer-reviewed CBMS 2003. The authors wish to acknowledge the experimental data and facilities provided by Jose Katz and Dan Petrylak in helping continuing tumor experiments with grant from Aventis Pharmaceuticals Pvt. Ltd.
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