International Journal of Radiation Biology
ISSN: 0955-3002 (Print) 1362-3095 (Online) Journal homepage: http://www.tandfonline.com/loi/irab20
A Potential Pitfall in the Use of Electroporation: Cellular Radiosensitization by Pulsed High-voltage Electric Fields C.M.L. West To cite this article: C.M.L. West (1992) A Potential Pitfall in the Use of Electroporation: Cellular Radiosensitization by Pulsed High-voltage Electric Fields, International Journal of Radiation Biology, 61:3, 329-334, DOI: 10.1080/09553009214551011 To link to this article: http://dx.doi.org/10.1080/09553009214551011
Published online: 03 Jul 2009.
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Date: 04 May 2016, At: 20:08
INT . J . RADIAT . BIOL .,
1992,
VOL .
61,
NO .
3, 329-334
A potential pitfall in the use of electroporation : cellular radiosensitization by pulsed high-voltage electric fields C . M . L . WESTt
Downloaded by [McMaster University] at 20:08 04 May 2016
(Received 21 June 1991 : revision received 16 September 1991 ; accepted 24 September 1991)
Abstract . Chinese hamster ovary cells have been exposed to high-voltage electric fields causing electroporation (EP) and the interaction between EP and radiation-induced cell lethality investigated . There was a voltage-dependent decrease in plating efficiency, assessed immediately following EP, and cell viability, assessed at 24 h . A linear decrease was seen for both . These decreases were accompanied by a voltage-dependent increase in cell volume, assessed immediately following EP. A good correlation between increases in cell volume and decreases in plating efficiency was seen (r=-0 . 91) . The application of electric fields immediately prior to, or following, irradiation led to a radiosensitization of the cells . This radiosensitization still occurred when a 6 h interval was left between radiation and EP but was lost when cells were irradiated 24 h prior to EP . When cells were irradiated following EP, the radiosensitization was lost with a 1 h interval between the two treatments . These results suggest that, when studying the combined cellular effects of EP of macromolecules and radiation, care should be taken that sufficient time has elapsed between the two modalities to prevent the radiosensitization of cells .
1. Introduction Electroporation (EP) is becoming increasingly popular as a method for the transfection of cells (Wong and Neumann 1982, Potter et al . 1984) . It is also an efficient method for introducing restriction endonucleases (RE) into cells (Winegar et al . 1989) . This is of interest as RE mimic radiation damage in their effects on DNA and can be used to examine the relationship between specific types of double-strand breaks (dsb) and their efficacy in inducing chromosomal aberrations and cell damage (reviewed by Bryant 1988) . There is interest in studying the radiosensitivity of cells following transfection with RE (and other macromolecules) . Fitzgerald et al . (1990) have transfected recombinant oncogenes into cells using EP and investigated their effect on radiosensitivity . Although not using EP, Tanzarella et al . (1990) have studied the interaction between X-ray- and REt Cancer Research Campaign Department of Experimental Radiation Oncology, Paterson Institute for Cancer Research, Christie Hospital, Manchester M20 9BX, UK .
induced lesions . In the course of studies aimed at investigating cell damage caused by a combination of electroporated RE and ionizing radiation, it was found that the EP procedure can alter the radiosensitivity of cells . 2.
Materials and methods
2.1 . Cell culture CHO cells were maintained as a monolayer culture in basal medium Eagle's (BME, Gibco, Paisley, Scotland) supplemented with 10% foetal calf serum (FCS, Advanced Protein Products, West Midlands, UK), penicillin (Gibco, 100 IU/ml) and streptomycin (Gibco, 0. 1 mg/ml), in a humidified 5% carbon dioxide atmosphere . Cells were harvested by incubation in 0-01% trypsin (180 IU/mg, Worthington Diagnostics Ltd, Freehold, New Jersey, USA) in 0.02% ethylenediamine tetra-acetic acid (BDH Chemicals Ltd, Poole, UK) made up in phosphate buffered saline (PBS, pH 7 .3). Cells were routinely tested to be Mycoplasma-free using the Hoechst fluorescence method. Exponentially growing cells were used in all experiments by seeding 1 .5 x 105 cells in 9 cm Petri dishes 3 days prior to use . 2.2. Electroporation A Bio-Rad Gene Pulser Transfection Apparatus was used for the EP of CHO cells . Following trypsinization, cells were washed twice in PBS and resuspended in PBS at a concentration of 5 x 10 6 cells/ml . Cells, 0 .8 ml, were then placed in disposable BioRad Gene Pulser Cuvettes with 0 .4 cm electrodes . EP was carried out at ambient temperature using the instrument settings recommended by the manufacturer for CHO cells . Gene Pulser voltages in the range 0 .5-2 . 0 kV, 25 microfarads capacitance and a time constant of 0 .7 ms were used . Immediately after EP the cells were placed in growth medium .
0020-7616/92 $3.00 © 1992 Taylor & Francis Ltd
330
C. M . L. West
Downloaded by [McMaster University] at 20:08 04 May 2016
2.3 . Irradiation and cell survival Cells were irradiated in medium plus 10% FCS or PBS, at room temperature, using a 137 Cs source with a dose-rate of 4.0 Gy/min . Following irradiation and/or EP cells were counted, diluted and appropriate numbers plated onto 60 mm Petri dishes . After 7-8 days incubation at 37°C in a humidified 5% carbon dioxide atmosphere, colonies were stained with gentian violet, and clonogenic cell survival determined . The plating efficiency (PE) of the cells was measured as the number of colonies formed divided by the number of cells plated and expressed as a percentage . The surviving fraction was measured as the number of colonies formed after treatment with radiation, EP or both, divided by the number of cells plated multiplied by PE expressed as a fraction . Three replicate plates were set up per cell density and two cell densities per point . Cell viability was assessed as the ability to exclude trypan blue . Radiation survival data were fitted to the linear quadratic equation using the program DRFIT run on a Micro VAX (Roberts 1990) .
and 50% of control levels (Andreason and Evans 1988) . As EP causes a transient increase in cellular permeability, either viability must be assessed 24h after applying EP or PE can be used . Both have been determined for CHO cells (Figure 1) . Following EP, a voltage-dependent decrease in cell viability, assessed at 24h, and PE assessed immediately, were seen . There was a general trend of lower values being obtained when cell viability was assessed, probably because both trypan blue exclusion and EP are predominantly membrane effects . There was a good correlation between increasing field strength and decreasing viability and plating efficiency (r= -0-97 for both) . These decreases were accompanied by a voltage-dependent increase in cell volume immediately following EP (Figure 2) . The increases in cell volume were well correlated (r=-0 . 91) with reductions in viability (Figure 3) . The irradiation of cells immediately following EP (10-15 min) led to an increase in the radiosensitivity of CHO cells (Figure 4) . This involved an increase in
a. 100-
2 .4 . Treatment with PvuII
The restriction endonuclease PvuII (Sigma) was introduced into cells immediately following irradiation with 8 Gy. PvuII, 100 units, was added to 0 . 8 ml of cells in PBS and the cells electroporated with 2 kV/0. 4 cm . The pellet technique was used also to introduce PvuII into cells. Cells, 1 .6 x 107, were centrifuged and washed in FCS . The supernatant was discarded and the cells resuspended in PvuII (7 pl, 100 units) . Following a 30 min incubation at 37°C, 10 ml ice-cold medium was added and the cells counted and plated as required. The time interval between irradiation and start of RE treatment was around 15 min .
80 T
60Cd 5 40200 b. 100
C
2.5. Cell volume Cell volumes were obtained using a Coulter Multisizer System (Coulter Electronics, Luton, UK . The median cell volume of a cell population was determined from the median channel number of the volume distribution and using a calibration constant derived from latex spheres of uniform size . 3. Results Maximum transfection following EP can be expected when cell viability falls to between 20%
Field strength(kV/0.4cm)
m rn
806040-
a 20 0 0
0.5 1 1 .5 Field strength(kV/0 .4cm)
2
Figure 1 . Decreasing cell viability assessed 24h following EP application (a) and plating efficiency assessed immediately (b) with increasing field strength . Points are the average± 1 SEM of four to 10 values .
Radiosensitization by electric fields
331
a.
a.
10 '-~
aX
x
10? x
C O
t
63
L
Field strength(kV/0 .4cm)
rn
b.
Downloaded by [McMaster University] at 20:08 04 May 2016
S
10
0
0 .5
1
1 .5
2
Field strength(kV/0 .4cm)
2
10 3
I
0
0.5
I
1
1 .5
2
Field strength(kV/0 .4cm)
Figure 2 . Cell diameter profiles for cells exposed to 2 kV and for a control population (a) . From such profiles values for median cell volume were obtained and plotted as a function of field strength (b) . Points are the data from five independent experiments represented by different symbols . The line through them has been fitted by eye .
100-
M
80T
= 60ae 4020 0
7
800
,
1
7
1200
1600
2000
Cell volume(pm3 )
Figure 3 . The relationship between cell volume and cell viability following the EP of CHO cells at different field strengths . Individual data points from four independent experiments have been plotted and a linear regression fitted to them.
Figure 4. Surviving fraction at 8 Gy as a function of field strength for CHO cells irradiated immediately (a) or 24 h (b) following EP . Different symbols represent independent experiments and the lines through them have been fitted by eye .
both a and fi (Figure 5) . Cells receiving irradiation only gave a survival curve with a = 0 .08 ± 0.03 Gy -1 and fl = 0 . 027 ± 0 .03 Gy - 2 . Those previously electroporated with 2 kV gave a survival curve with a=0 . 41±0 . 04Gy -1 and P=0. 016±0 . 04Gy -2 . The two curves were highly significantly different (p
ISSN: 0955-3002 (Print) 1362-3095 (Online) Journal homepage: http://www.tandfonline.com/loi/irab20
A Potential Pitfall in the Use of Electroporation: Cellular Radiosensitization by Pulsed High-voltage Electric Fields C.M.L. West To cite this article: C.M.L. West (1992) A Potential Pitfall in the Use of Electroporation: Cellular Radiosensitization by Pulsed High-voltage Electric Fields, International Journal of Radiation Biology, 61:3, 329-334, DOI: 10.1080/09553009214551011 To link to this article: http://dx.doi.org/10.1080/09553009214551011
Published online: 03 Jul 2009.
Submit your article to this journal
Article views: 3
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=irab20 Download by: [McMaster University]
Date: 04 May 2016, At: 20:08
INT . J . RADIAT . BIOL .,
1992,
VOL .
61,
NO .
3, 329-334
A potential pitfall in the use of electroporation : cellular radiosensitization by pulsed high-voltage electric fields C . M . L . WESTt
Downloaded by [McMaster University] at 20:08 04 May 2016
(Received 21 June 1991 : revision received 16 September 1991 ; accepted 24 September 1991)
Abstract . Chinese hamster ovary cells have been exposed to high-voltage electric fields causing electroporation (EP) and the interaction between EP and radiation-induced cell lethality investigated . There was a voltage-dependent decrease in plating efficiency, assessed immediately following EP, and cell viability, assessed at 24 h . A linear decrease was seen for both . These decreases were accompanied by a voltage-dependent increase in cell volume, assessed immediately following EP. A good correlation between increases in cell volume and decreases in plating efficiency was seen (r=-0 . 91) . The application of electric fields immediately prior to, or following, irradiation led to a radiosensitization of the cells . This radiosensitization still occurred when a 6 h interval was left between radiation and EP but was lost when cells were irradiated 24 h prior to EP . When cells were irradiated following EP, the radiosensitization was lost with a 1 h interval between the two treatments . These results suggest that, when studying the combined cellular effects of EP of macromolecules and radiation, care should be taken that sufficient time has elapsed between the two modalities to prevent the radiosensitization of cells .
1. Introduction Electroporation (EP) is becoming increasingly popular as a method for the transfection of cells (Wong and Neumann 1982, Potter et al . 1984) . It is also an efficient method for introducing restriction endonucleases (RE) into cells (Winegar et al . 1989) . This is of interest as RE mimic radiation damage in their effects on DNA and can be used to examine the relationship between specific types of double-strand breaks (dsb) and their efficacy in inducing chromosomal aberrations and cell damage (reviewed by Bryant 1988) . There is interest in studying the radiosensitivity of cells following transfection with RE (and other macromolecules) . Fitzgerald et al . (1990) have transfected recombinant oncogenes into cells using EP and investigated their effect on radiosensitivity . Although not using EP, Tanzarella et al . (1990) have studied the interaction between X-ray- and REt Cancer Research Campaign Department of Experimental Radiation Oncology, Paterson Institute for Cancer Research, Christie Hospital, Manchester M20 9BX, UK .
induced lesions . In the course of studies aimed at investigating cell damage caused by a combination of electroporated RE and ionizing radiation, it was found that the EP procedure can alter the radiosensitivity of cells . 2.
Materials and methods
2.1 . Cell culture CHO cells were maintained as a monolayer culture in basal medium Eagle's (BME, Gibco, Paisley, Scotland) supplemented with 10% foetal calf serum (FCS, Advanced Protein Products, West Midlands, UK), penicillin (Gibco, 100 IU/ml) and streptomycin (Gibco, 0. 1 mg/ml), in a humidified 5% carbon dioxide atmosphere . Cells were harvested by incubation in 0-01% trypsin (180 IU/mg, Worthington Diagnostics Ltd, Freehold, New Jersey, USA) in 0.02% ethylenediamine tetra-acetic acid (BDH Chemicals Ltd, Poole, UK) made up in phosphate buffered saline (PBS, pH 7 .3). Cells were routinely tested to be Mycoplasma-free using the Hoechst fluorescence method. Exponentially growing cells were used in all experiments by seeding 1 .5 x 105 cells in 9 cm Petri dishes 3 days prior to use . 2.2. Electroporation A Bio-Rad Gene Pulser Transfection Apparatus was used for the EP of CHO cells . Following trypsinization, cells were washed twice in PBS and resuspended in PBS at a concentration of 5 x 10 6 cells/ml . Cells, 0 .8 ml, were then placed in disposable BioRad Gene Pulser Cuvettes with 0 .4 cm electrodes . EP was carried out at ambient temperature using the instrument settings recommended by the manufacturer for CHO cells . Gene Pulser voltages in the range 0 .5-2 . 0 kV, 25 microfarads capacitance and a time constant of 0 .7 ms were used . Immediately after EP the cells were placed in growth medium .
0020-7616/92 $3.00 © 1992 Taylor & Francis Ltd
330
C. M . L. West
Downloaded by [McMaster University] at 20:08 04 May 2016
2.3 . Irradiation and cell survival Cells were irradiated in medium plus 10% FCS or PBS, at room temperature, using a 137 Cs source with a dose-rate of 4.0 Gy/min . Following irradiation and/or EP cells were counted, diluted and appropriate numbers plated onto 60 mm Petri dishes . After 7-8 days incubation at 37°C in a humidified 5% carbon dioxide atmosphere, colonies were stained with gentian violet, and clonogenic cell survival determined . The plating efficiency (PE) of the cells was measured as the number of colonies formed divided by the number of cells plated and expressed as a percentage . The surviving fraction was measured as the number of colonies formed after treatment with radiation, EP or both, divided by the number of cells plated multiplied by PE expressed as a fraction . Three replicate plates were set up per cell density and two cell densities per point . Cell viability was assessed as the ability to exclude trypan blue . Radiation survival data were fitted to the linear quadratic equation using the program DRFIT run on a Micro VAX (Roberts 1990) .
and 50% of control levels (Andreason and Evans 1988) . As EP causes a transient increase in cellular permeability, either viability must be assessed 24h after applying EP or PE can be used . Both have been determined for CHO cells (Figure 1) . Following EP, a voltage-dependent decrease in cell viability, assessed at 24h, and PE assessed immediately, were seen . There was a general trend of lower values being obtained when cell viability was assessed, probably because both trypan blue exclusion and EP are predominantly membrane effects . There was a good correlation between increasing field strength and decreasing viability and plating efficiency (r= -0-97 for both) . These decreases were accompanied by a voltage-dependent increase in cell volume immediately following EP (Figure 2) . The increases in cell volume were well correlated (r=-0 . 91) with reductions in viability (Figure 3) . The irradiation of cells immediately following EP (10-15 min) led to an increase in the radiosensitivity of CHO cells (Figure 4) . This involved an increase in
a. 100-
2 .4 . Treatment with PvuII
The restriction endonuclease PvuII (Sigma) was introduced into cells immediately following irradiation with 8 Gy. PvuII, 100 units, was added to 0 . 8 ml of cells in PBS and the cells electroporated with 2 kV/0. 4 cm . The pellet technique was used also to introduce PvuII into cells. Cells, 1 .6 x 107, were centrifuged and washed in FCS . The supernatant was discarded and the cells resuspended in PvuII (7 pl, 100 units) . Following a 30 min incubation at 37°C, 10 ml ice-cold medium was added and the cells counted and plated as required. The time interval between irradiation and start of RE treatment was around 15 min .
80 T
60Cd 5 40200 b. 100
C
2.5. Cell volume Cell volumes were obtained using a Coulter Multisizer System (Coulter Electronics, Luton, UK . The median cell volume of a cell population was determined from the median channel number of the volume distribution and using a calibration constant derived from latex spheres of uniform size . 3. Results Maximum transfection following EP can be expected when cell viability falls to between 20%
Field strength(kV/0.4cm)
m rn
806040-
a 20 0 0
0.5 1 1 .5 Field strength(kV/0 .4cm)
2
Figure 1 . Decreasing cell viability assessed 24h following EP application (a) and plating efficiency assessed immediately (b) with increasing field strength . Points are the average± 1 SEM of four to 10 values .
Radiosensitization by electric fields
331
a.
a.
10 '-~
aX
x
10? x
C O
t
63
L
Field strength(kV/0 .4cm)
rn
b.
Downloaded by [McMaster University] at 20:08 04 May 2016
S
10
0
0 .5
1
1 .5
2
Field strength(kV/0 .4cm)
2
10 3
I
0
0.5
I
1
1 .5
2
Field strength(kV/0 .4cm)
Figure 2 . Cell diameter profiles for cells exposed to 2 kV and for a control population (a) . From such profiles values for median cell volume were obtained and plotted as a function of field strength (b) . Points are the data from five independent experiments represented by different symbols . The line through them has been fitted by eye .
100-
M
80T
= 60ae 4020 0
7
800
,
1
7
1200
1600
2000
Cell volume(pm3 )
Figure 3 . The relationship between cell volume and cell viability following the EP of CHO cells at different field strengths . Individual data points from four independent experiments have been plotted and a linear regression fitted to them.
Figure 4. Surviving fraction at 8 Gy as a function of field strength for CHO cells irradiated immediately (a) or 24 h (b) following EP . Different symbols represent independent experiments and the lines through them have been fitted by eye .
both a and fi (Figure 5) . Cells receiving irradiation only gave a survival curve with a = 0 .08 ± 0.03 Gy -1 and fl = 0 . 027 ± 0 .03 Gy - 2 . Those previously electroporated with 2 kV gave a survival curve with a=0 . 41±0 . 04Gy -1 and P=0. 016±0 . 04Gy -2 . The two curves were highly significantly different (p
