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Effect of mitomycin C on the uptake of photofrin II in a human colon adenocarcinoma cell line
Cancer Letters, 64 (1992) 155- 162 Elsevier Scientific Publishers Ireland Ltd.
155
Effect of mitomycin C on the uptake of photofrin II in a human colon adenocarcinoma cell line L.W.
Ma, J. Moan, H.B. Steen,
Department
of Biophysics,
K. Berg and Q. Peng
The Institute for Cancer Research,
Montebello,
0310
Oslo 3 (Norway)
(Received 7 January 1’992) (Revision received 13 April 1992) (Accepted 13 April 1992)
Keywords: photodynamic therapy; frin II; mitomycin C; flow cytometry
Summary Now cytometry (KM) was used to investigate the effect of mitomycin C (MC) on the cellular uptake of Photofrin II (PII) in a cultured human colon adenocarcinoma cell line (WiDr). The surface area of the cells increased as they passed through the cell cycle from Go/G1 to Gs/M phase. MC retarded the cells in G/M phase and enhanced the surface area of the cells. A 1.3-2.9fold increase in the cell surface area and a 1.3 - 2.7-fold increase in the cellular uptake of PI1 in the tumor cells was observed after 2 h -8 h incubation with MC. Within each sample, an almost linear relationship between the intensity of PU fluorescence in the cells and the surface area of the cells was found. However, for the cells incubated with MC the surface area was not the only determinant of PII uptake. Effects of MC on the cell cycle, the cell surface area and the permeability of the cell membrane are suggested as possible reasons for the increase of cellular uptake of P/l in the tumor cells.
Correspondence to: L.W. Ma, Department of Biophysics, The Institute for Cancer Rese,srch, Montebello, 0310 0sl0 3, Norway. Abbreuiations: PDT, photodynamic HPD, hematoporphyrin derivative; Adriamycin; FCM, flow cytometry. 0304-3835/92/$05.00 Printed and Published
therapy; PII, photofrin 11; MC, mitomycin C; ADM,
@ 1992 Elsevier Scientific Publishers in Ireland
photo-
Introduction Effects of photodynamic therapy (PDT) on tumors in combination with chemotherapy have been studied in several investigations [ 1 - 61. Different mechanisms have been proposed to explain the synergistic effects found in some of these studies. However, little data is available regarding the effect of chemotherapeutic drugs on the cellular uptake of photosensitizers. The study of Cowled et al. [l] showed that Adriamycin (ADM) and methotrexate increased the tumor uptake of hematoporphyrin (HPD), while in vitro ADM reduced the cellular uptake of HPD and methotrexate had no effect on the uptake of HPD. The administration of methylprednisolone acetate concurrently with HPD to mice bearing Lewis lung carcinoma markedly reduced the intensity of HPD fluorescence in the tumor and inhibited the response of the tumor to PDT [3]. Mitomycin C (MC) has been described as an effective antitumor agent for clinical use [7]. The effects of MC and ionizing radiation on tumors in vivo have been shown to be additive [8]. Our preliminary work has indicated that MC can potentiate PDT in vitro (Ma et al., unpublished data). The present study was underIreland Ltd
156
taken to explore whether MC has influence on the cellular uptake of PII. Materials
any
and Methods
Cell culture The established cell line WiDr, derived from a primary adenocarcinoma of the rectosigmoid colon [9], was used throughout. The cells were maintained in exponential growth in RPM1 1640 medium with 10% fetal calf serum (FCS) , 100 units/ml penicillin and 100 pg/ml streptomycin. Cultures were incubated in a humidified 5% COP atmosphere and subcultured twice a week using 0.01% trypsin in 0.02% EDTA. Drug exposure and drug uptake Photofrin 11was provided by Quadra Logic Technologies (Vancouver, Canada). A single batch was used for all experiments and small aliquots were stored at - 20°C and thawed to room temperature immediately prior to experiments. MC was obtained from Medac, GmbH (Hamburg, Germany). A 2-mg quantity of drug powder was reconstituted in 5 ml of 0.9% saline and appropriate drug dilutions were made with growth medium. Exponentially growing cells (8 h post inoculation) were exposed to 0.1 pg/ml MC in RPM1 1640 medium plus 10% FCS for different times, washed twice with PBS and allowed to grow in fresh medium without drug. Twenty four hours after the initiation of MC treatment the cells were incubated with PI1 (10 pg/ml) in the medium with 10% FCS for 16 h. After this incubation, the cells were rinsed three times with ice-cold PBS and trypsinized to single cell suspensions for flow cytometry measurements. Cell suruiual Cell survival
was
by
the
HBOlOO). Fluorescence emission passed through a 410-nm long pass filter. For the measurements of PI1 uptake, the cells were harvested at 16 h after the addition. of PII. PI1 fluorescence was excited with 436 nm light and fluorescence emission measured using a 620-nm long pass filter. For each experiment, two parallel samples were prepared simultaneously. One of the them was used for the analysis of DNA content, the other one was used for the measurement of PI1 fluorescence. A minimum of 20 000 cells were measured for each analysis. Results Figure 1 shows that at a concentration of 0.1 pg/ml MC is cytotoxic and drastically reduces the ability of the cells to divide and form colonies when applied for more than 4 h. Therefore, most of the present experiments were performed with 4 h incubation of the cells with MC. Figure 2A depicts the biparametric histogram of low angle light scatter (LSl) versus blue fluorescence (FLl) of cells stained with Hoechst 33258. LSl is proportional to cell surface area, whereas FL1 is a measure of DNA content. Hence, this histogram shows the cell surface area increased through all phases of the cell cycle: Go/G1 < S < G2/M. The
157
Exposure timer, of cells to MC (hours) 16 I2 4 a
1 \
i\y
Fig. 1. Survival curve (colony formation assay) for cells exposed to 0.1 &ml MC for 2 h, 4 h, 8 h and 16 h, respectively. Bars: S.D. from the three independent experiments.
biparametric histogram of LSl vs. red fluorescence (FLZ) (Fig. 2B) related to intracellular PI1 shows that the PII fluorescence was roughly proportional to the cell surface area.
0
FL1
0
FL2
Fig. 2. Dual parameter (fluorescence/low angle light scatter) histogram of WliDr cells. (A), ordinate (LSl) represents the signal of low angle light scatter, abscissa (FLl) represents the intensity of fluorescence of the DNA specific dye HO 33258. (B), ordinate (LSl) is the same as in (A), abscissa (FL2) represents the intensity of PI1 fluorescence.
The DNA and light scattering histograms of Fig. 3 show that on increasing the exposure time of cells to 0.1 fig/ml MC from 2 h to 8 h, there was a large increase in the percentage of cells in the GJM phase as well as an increase in the cell surface area. This increase was accompanied by a significant enhancement of the cellular uptake of PII, as seen by a marked shifting of the PII fluorescence histograms to the right. PI1 alone, at the concentrations used here, had no effect on the cell cycle. Cells incubated with both MC and PI1 exhibited a 1.3-3.5-fold increase in the number of G,JM phase cells, a 1.3 -2.3-fold increase in cell surface area and a 1.3 - 2.7-fold increase in the cellular fluorescence of PI1 as compared to control cells incubated with only PII (Table I). The effects of MC on cellular PI1 uptake changed with the time interval between the removal of MC and the incubation with PI1 (Fig. 4). The increase of the fluorescence of PI1 was larger when PI1 was given at 20 h after a 4-h incubation with 0.1 pg/ml MC than when PI1 was added at 44 h or 68 h after such incubation with MC. With increasing time after exposure to MC, the changes in DNA content, cell surface area and PI1 fluorescence can recover to the level of cells treated with PI1 only. In Fig. 4, MC clearly induced an accumulation of the cells in GJM phase. The fraction of cells in GJM phase (about 55%) 20 h after incubation with MC is apparently larger than the number of cells killed by MC (20%) (Fig. 1). This indicates that a large fraction of the cells is only transiently blocked in GJM phase and will divide at a later point. Hence, it appears that most of the affected cells were recovering after exposure to MC rather than being replaced by normal cells. In order to determine the effect of dead cells on the experimental data, after the treatments of MC and PII mentioned above, the cells were incubated with propidium iodide, which is staining only for dead cells, before FCM measurement. In each sample a small proportion of dead cells (lo- 15%) was observed. The PI1 fluorescence of the dead cells was
158
DNA Histogram
CeII Surface Area PU Fluorescence
Pll only
FL1
2h MC,Pll
K
4 h MC, PI1
FL1
8h MC,Pll
l-/blLA
Fig. 3. PI1 (FL2), DNA (FLl) and cell surface area (LSl) histograms of cells which were exposed to 0.1 pg/ml MC for 2 h, 4 h and 8 h, then washed and incubated further in pure medium with 10%. FCS for 22 h, 20 h and 16 h, respectively, before they were incubated with PI1 (10 pg/ml) for 16 h. Abscissa unit is channel number (relative linear scale) representing, respectively, the intensity of fluorescence of the DNA specific dye HO 33258 (DNA histograms, FLl); the size of cells (histograms of cell surface area, LSl); the intensity of intracellular PII fluorescence (histograms of PII fluorescence, FL2). All ordinates represent the number of cells per channel (relative linear scale).
somewhat larger than that of vital cells, but this difference was not sufficient to explain the increase of the PII fluorescence of the total samples. Figure 5 shows an approximately linear rela-
tionship between the fluorescence of PII and the ceil surface area as measured by light scattering. The slope of the curves for MC-treated ce!ls was somewhat larger than for the control, which is to say that the PI1 fluorescence of cells
159
Table I. The effects of mitomycin cellular fluorescence of PII. Sample
PII (control) 2 h MC, 22 4 h MC, 20 8 h MC, 16 4 h MC, 44
h h h h
‘PM, PM, PM, PM,
16 h PII 16 h PII 16 h PI1 161 h PI1
4 h MC, 68 h PM, 16 h PI1
C on the percentage
of cells in S and Gs/M phase,
cell surface area and average
% Cells in S and GJM
Cell surface area (rel. units)
PII fluorescence (rel. units)
30 36 27 24 43
19 33 56 66 25
1 1.3 1.6 2.3 1.3
37
17
1.1
1 1.3 1.8 2.7 1.7 1.1
The cells were incubated with 0.1 rg/ml MC for 2 h, 4 h and 8 h, respectively. PI1 (10 pg/ml) was given for 16 h starting 16 - 68 h after removal of MC. Each value is from median of 8000 cells analyzed by fIow cytometry. ‘PM: pure medium with 10% FCS.
DNA Histogram
Ceil Surface Area Pll fluorescence
FL1
PI1 only
‘IL I !&LQ_ 4 I i
IS1
FL1
4h MC,20h PM*,Pll
FL1
4 h MC, 44 h PM, F’ll
FLI
4h MC,68h PM,Pll
J,
,h__
Fig. 4. Histograms of PI1 fluorescence (FL2), DNA content (FLl) and surface area (LSl) of cells which were incubated with 10 fig/ml PI1 for 16 h starting at 20 h, 44 h and 68 h, respectively, after removal of 0.1 rg/ml MC to which the cells were exposed for 4, h. The units of ordinate and abscissa are same as in Fig. 3. * PM, pure medium with 10% FCS.
160
250
E
200
6 0 z
150
6 3 z
100
a 50
,L
0
0
I
1
60
120
Cel
I
.
Surface
.
I
I
160
240
Area
Fig. 5. Cellular PI1 fluorescence as a function of cell surface area (LSl). Symbols: 0 control cells incubated with PII for 16 h; x cells were exposed to MC for 2 h. washed and incubated further in pure medium with 10% FCS for 22 h and then PII was given for 16 h; 0 cells exposed to MC for 4 h, washed and incubated in pure medium with 10% FCS for 20 h and then PI1 was added for 16 h; A 8 h, MC, washing and 16 h, pure medium with 10% FCS and then PI1 16 h; + 4 h, MC, washing and 44 h, pure medium with 10% FCS, followed by PI1 16 h; 0 4 h, MC, washing and 68 h, pure medium with 10% FCS and then PII 16 h. Each point represents approximately 500 - 1000 cells. MC concentration: 0.1 j.kg/ml, PII concentration: 10 pg/ml.
exposed to MC was higher than that of control cells of equal size. Thus, it seems that the effect of MC on PI1 uptake was related not only to cell surface area, but also to other factors, such as the permeability of the cell membrane (see Discussion).
Discussion Mitomycin C is an antitumor antibiotic with a bifunctional alkylating action. It has been used clinically for treating gastrointestinal and
other cancers [ 121. Its mode of action is believed to be mainly the formation of cross-links of DNA, retardation of the cell cycle and inhibition of cell growth [ 131. The cytotoxicity of MC is dependent on drug concentration and exposure time (Ma et al, unpublished data). The concentration applied in the present work is clinically relevant since Shimizu et al. [15] found an MC concentration of 0.1 pg/ml in plasma 1 h after a bronchial artery infusion of 20 mg MC in cancer patients. The fluorescence of PI1 in the WiDr ceils increased with increasing exposure time to MC implying that MC facilitates the cellular uptake of PII. Possible mechanisms of the effect of MC on the cellular uptake of PI1 are considered below. Cell surface area is a major determinant of the cellular uptake of PI1 (Figs. 2 and 5). Our data demonstrate that the surface area of WiDr cells increases throughout all parts of interphase (Fig. 2). The early study of Steen et al. [16] showed that the size of synchronized NHIK 3025 cells increased approximately linearly with time through the cell cycle. MC can enhance the cell surface area by retarding the cells in Gz/M phase (Fig. 3). A nearly linear correlation between the fluorescence intensity of intracellular PI1 and the cell surface area was found in Fig. 5. This finding is in agreement with the report of Christensen et al., who found that the amount of cell bound hematoporphyrin increased as cells proceeded through the cell cycle and was approximately doubled from Gl to late G2 [17]. The mechanisms by which PI1 is taken up by cells are not well known. PII, which consists primarily of lipophilic components, localizes mainly in cell membrane structures [18]. If the first step of passage into the cell is that the dye is passively dissolved in the cell membrane, the observed proportionality between the cellular uptake of PI1 and the cell surface area is to be expected. It has been suggested that LDL receptors on the cell membrane may play a role in cellular uptake of PI1 [19]. A significant fraction of PI1 binds to LDL in serum [20]. Again, it seems likely that the number of such
161
receptors is proportional to the cell surface area. Bare1 et al. [21] proposed that there may be a lower number of LDL receptors in small cells. The present finding that small cells take up relatively little of PI1 is consistent with the work of West et al. [22]. These authors reported that with increasing spheroid size of WiDr cells, there was a decrease in intracellular HPD levels, because the larger spheroids have an increased proportion of Gl-like cells with smaller volume [23]. However, it appears that cell surface area is not the only determinant of the effect of MC on the cellular uptake of PI1 (Fig. 5). Other factors, like membrane permeability may be of importance. MC has been shown to be capable of generating oxygen free radicals which produce substantial (damage to biologic membranes [24]. Therefore, one may speculate that the permeability of the cell membrane is increased probably by MC. Thus, a passive drug entry of PII into the cell (independent of a facilitated transportation by LDL receptors) may be enhanced by a toxic effect of MC on the cell membrane. Further experiments are needed to elucidate the nature of possible interactions between MC and the cell membrane. It is not clear whether the increase of intracellular PI1 induced by the effects of MC on the cell cycle is specific for tumor cells. However, the fact that tumor tissues of patients with gastric and colorectal cancers are more sensitive to MC than are adjacent normal tissues has been demonstrated [25]. They proposed that since the DNA synthesis and replication are more active in tumor tissues than in normal tissues, tumor tissues are more likely to be affected by a DNA-interacting drug such as MC. It remains to be seen whether MC enhances the uptake of PII in tumors with some selectivity.
assistance, Magne Kongshaug for valuable discussion and Professor Claude Rimington for critical reading of the manuscript. References 1
2
3
4
5
6
7
8
9
10
11
12
13
Mackenzie, L. and Forbes, I.J. (1987) modulation of photodynamic therapy
with hematoporphyrin derivative and light. Cancer Res., 47, 971-974. Creekmore, S.P. and Zaharko, D.S. (1983) Modification of chemotherapeutic effects on L 1210 cells using hematoporphyrin
Acknowledgements The present work was supported by The Association for International Cancer Research. We thank Hilde Saether for technical
Cowled, P.A., Pharmacological
and
light.
Cancer
Res.,
43,
5252 - 5257. Cowled, P.A., Mackenzie, L. and Forbes, I.J. (1985) Potentiation of photodynamic therapy with haematoporphyrin derivatives by glucocorticoids. Cancer Lett., 29, 107- 114. Edell, ES. and Cortese, D.A. (1988) Combined effects of hematoporphyrin derivative phototherapy and adriamycin in a murine tumor mode. Lasers Surg. Med., 8, 413-417. Nahabedian, M.Y., Cohen, R.A., Contino, M.F., Terem, T.M., Wright, W.H., Berns, M.W. and Wile, A.G. (1988) Combination cytotoxic chemotherapy with cisplatin or doxorubicin and photodynamic therapy in murine tumors, J. Natl. Cancer Inst., 80, 738-743. Kessel, D. (1977) Effects of photoactivated porphyrins at the cell surface of leukemia L 1210 cells. Biochemistry, 16, 3443-3449. Sartorelli, A.C. (1986) The role of mitomycin antibiotics in the chemotherapy of solid tumors. Biochem. Pharmacol., 35, 67-69. Holden, S.A., Herman, T.S. and Teicher, B.A. (1990) Addition of a hypoxic cell selective cytotoxic agent (mitomycin C or porfiromycin) to Fluosol-DA/carbogen/radiation. Radiother. Oncol., 18, 59 - 70. Noguchi, P., Wallace, R., Johnson, J., Earley, E.M., O’Brien, S., Ferrone, S., Pellegrino, M.A., Milstein, J., Needy, C., Browne, W. and Petricciani, J. (1979) Characterization of WiDr: a human colon carcinoma cell line. In Vitro, 15, 401-408. Berg, K. and Moan, J. (1988) Photodynamic effects of photofrin II on cell division in human NHIK 3025 cells. Int. J. Radiat. Biol., 53, 797-811. Steen, H.B. (1986) Simultaneous separate detection of low angle and large a,tigle light scattering in an arc lampbased flow cytometer. Cytometry, 7, 445 -449. Moore, G.E., Bross, I.D.J., Ausman, R., Nadler, S., Jones, R., Jr., Slack, N. and Rimm, A.A. (1968) Effects of mitomycin C (NSC-26980) in 346 patients with advanced cancer. Cancer Chemother. Rep. Part I., 52, 678-684. Goldberg, I.H. (1965) Mode of action of antibiotics. II. Drugs affecting nucleic acid and protefn synthesis. Am. J. Med., 39, 722-752.
162
14
15
16
17
18
19
20
Barlogie, B. and Drewinko, B. (1980) Lethal and cytokinetic effects of mitomycin C on cultured human colon cancer cells. Cancer Res., 40, 1973- 1980. Shimizu, E., Nakamura, Y., Mukai, J.N., Tani, K.. Yamashita, T., Hoko, F., Hashimoto, Y. and Ogura, T. (1991) Pharmacokinetics of bronchial artery infusion of mitomycin in patients with non-small cell lung cancer. Eur. J. Cancer, 27, 1046- 1048. Steen, H.B. and Lindmo, T. (1978) Cellular and nuclear volume during the cell cycle of NHIK 3025 cells. Cell Tiss. Kinet., 11, 69-81. Christensen, T. and Moan, J. (1980) Binding of hematoporphyrin to synchronized cells from the line NHIK 3025. Cancer Lett., 9, 105- 110. Moan, J., Mcghie, J.B. and Christensen, T. (1982) Hematoporphyrin derivative: photosensitizing efficiency and cellular uptake of its components. Photobiochem. Photobiophys., 4, 337 - 345. Candide, C., Morliere, P., Maziere, J.C., Goldstein, S., Santus, R., Dubertret, L., Reyftmann, J.P. and Polonovski, J. (1986) In vitro interaction of the photoactive anticancer porphyrin derivative photofrin II with low density lipoprotein, and its delivery to cultured human fibroblasts. FEBS Lett., 207, 133- 138. Kongshaug,
M., Moan,
J. and Brown,
S.B.
(1989) The
21
distribution of porphyrins with different tumor localising ability among human plasma proteins. Br. J. Cancer., 59, 184 - 188. Barel, A., Jori, G., Perin, A., Romandini, P., Pagnan, A. and Biffanti, S. (1986) Role of high-, low- and very-lowdensity lipoproteins in the transport and delivery of hematoporphyrin in vivo. Cancer Lett., 32, 145- 155.
22
West, C.M.L. and Moore, J.V. (1989) Flow cytometric analysis of intracellular hematoporphyrin derivative in human tumor cells and multicellular spheroids. Photochem. Photobiol., 50, 665 - 669.
23
West, C.M.L. and Sutherland, R.M. (1987) The radiation response of a human colon adenocarcinoma grown in monolayer, as spheroids and in nude mice. Radiat. Res., 112, 105- 115.
24
Trush, M.A., Mimnaugh, E.G., Ginsburg, E. and Gram, T.E. (1982) Studies on the in vitro interaction of mitomycin C, nitrofurantoin and paraquat with pulmonary microsomes. Stimulation of reactive oxygen-dependent lipid peroxidation. Biochem. Pharmacol., 31. 805 - 814. Maehara, Y., Kusumoto, H., Kusumoto, T., Anai, H. and Sugimachi, K. (1989) Tumor tissue is more sensitive to mitomycin C, carboquone and aclacinomycin A than is ad-
25
jacent normal
tissue in vitro. J. Surg.
Oncol.,
40, 4-
7.
155
Effect of mitomycin C on the uptake of photofrin II in a human colon adenocarcinoma cell line L.W.
Ma, J. Moan, H.B. Steen,
Department
of Biophysics,
K. Berg and Q. Peng
The Institute for Cancer Research,
Montebello,
0310
Oslo 3 (Norway)
(Received 7 January 1’992) (Revision received 13 April 1992) (Accepted 13 April 1992)
Keywords: photodynamic therapy; frin II; mitomycin C; flow cytometry
Summary Now cytometry (KM) was used to investigate the effect of mitomycin C (MC) on the cellular uptake of Photofrin II (PII) in a cultured human colon adenocarcinoma cell line (WiDr). The surface area of the cells increased as they passed through the cell cycle from Go/G1 to Gs/M phase. MC retarded the cells in G/M phase and enhanced the surface area of the cells. A 1.3-2.9fold increase in the cell surface area and a 1.3 - 2.7-fold increase in the cellular uptake of PI1 in the tumor cells was observed after 2 h -8 h incubation with MC. Within each sample, an almost linear relationship between the intensity of PU fluorescence in the cells and the surface area of the cells was found. However, for the cells incubated with MC the surface area was not the only determinant of PII uptake. Effects of MC on the cell cycle, the cell surface area and the permeability of the cell membrane are suggested as possible reasons for the increase of cellular uptake of P/l in the tumor cells.
Correspondence to: L.W. Ma, Department of Biophysics, The Institute for Cancer Rese,srch, Montebello, 0310 0sl0 3, Norway. Abbreuiations: PDT, photodynamic HPD, hematoporphyrin derivative; Adriamycin; FCM, flow cytometry. 0304-3835/92/$05.00 Printed and Published
therapy; PII, photofrin 11; MC, mitomycin C; ADM,
@ 1992 Elsevier Scientific Publishers in Ireland
photo-
Introduction Effects of photodynamic therapy (PDT) on tumors in combination with chemotherapy have been studied in several investigations [ 1 - 61. Different mechanisms have been proposed to explain the synergistic effects found in some of these studies. However, little data is available regarding the effect of chemotherapeutic drugs on the cellular uptake of photosensitizers. The study of Cowled et al. [l] showed that Adriamycin (ADM) and methotrexate increased the tumor uptake of hematoporphyrin (HPD), while in vitro ADM reduced the cellular uptake of HPD and methotrexate had no effect on the uptake of HPD. The administration of methylprednisolone acetate concurrently with HPD to mice bearing Lewis lung carcinoma markedly reduced the intensity of HPD fluorescence in the tumor and inhibited the response of the tumor to PDT [3]. Mitomycin C (MC) has been described as an effective antitumor agent for clinical use [7]. The effects of MC and ionizing radiation on tumors in vivo have been shown to be additive [8]. Our preliminary work has indicated that MC can potentiate PDT in vitro (Ma et al., unpublished data). The present study was underIreland Ltd
156
taken to explore whether MC has influence on the cellular uptake of PII. Materials
any
and Methods
Cell culture The established cell line WiDr, derived from a primary adenocarcinoma of the rectosigmoid colon [9], was used throughout. The cells were maintained in exponential growth in RPM1 1640 medium with 10% fetal calf serum (FCS) , 100 units/ml penicillin and 100 pg/ml streptomycin. Cultures were incubated in a humidified 5% COP atmosphere and subcultured twice a week using 0.01% trypsin in 0.02% EDTA. Drug exposure and drug uptake Photofrin 11was provided by Quadra Logic Technologies (Vancouver, Canada). A single batch was used for all experiments and small aliquots were stored at - 20°C and thawed to room temperature immediately prior to experiments. MC was obtained from Medac, GmbH (Hamburg, Germany). A 2-mg quantity of drug powder was reconstituted in 5 ml of 0.9% saline and appropriate drug dilutions were made with growth medium. Exponentially growing cells (8 h post inoculation) were exposed to 0.1 pg/ml MC in RPM1 1640 medium plus 10% FCS for different times, washed twice with PBS and allowed to grow in fresh medium without drug. Twenty four hours after the initiation of MC treatment the cells were incubated with PI1 (10 pg/ml) in the medium with 10% FCS for 16 h. After this incubation, the cells were rinsed three times with ice-cold PBS and trypsinized to single cell suspensions for flow cytometry measurements. Cell suruiual Cell survival
was
by
the
HBOlOO). Fluorescence emission passed through a 410-nm long pass filter. For the measurements of PI1 uptake, the cells were harvested at 16 h after the addition. of PII. PI1 fluorescence was excited with 436 nm light and fluorescence emission measured using a 620-nm long pass filter. For each experiment, two parallel samples were prepared simultaneously. One of the them was used for the analysis of DNA content, the other one was used for the measurement of PI1 fluorescence. A minimum of 20 000 cells were measured for each analysis. Results Figure 1 shows that at a concentration of 0.1 pg/ml MC is cytotoxic and drastically reduces the ability of the cells to divide and form colonies when applied for more than 4 h. Therefore, most of the present experiments were performed with 4 h incubation of the cells with MC. Figure 2A depicts the biparametric histogram of low angle light scatter (LSl) versus blue fluorescence (FLl) of cells stained with Hoechst 33258. LSl is proportional to cell surface area, whereas FL1 is a measure of DNA content. Hence, this histogram shows the cell surface area increased through all phases of the cell cycle: Go/G1 < S < G2/M. The
157
Exposure timer, of cells to MC (hours) 16 I2 4 a
1 \
i\y
Fig. 1. Survival curve (colony formation assay) for cells exposed to 0.1 &ml MC for 2 h, 4 h, 8 h and 16 h, respectively. Bars: S.D. from the three independent experiments.
biparametric histogram of LSl vs. red fluorescence (FLZ) (Fig. 2B) related to intracellular PI1 shows that the PII fluorescence was roughly proportional to the cell surface area.
0
FL1
0
FL2
Fig. 2. Dual parameter (fluorescence/low angle light scatter) histogram of WliDr cells. (A), ordinate (LSl) represents the signal of low angle light scatter, abscissa (FLl) represents the intensity of fluorescence of the DNA specific dye HO 33258. (B), ordinate (LSl) is the same as in (A), abscissa (FL2) represents the intensity of PI1 fluorescence.
The DNA and light scattering histograms of Fig. 3 show that on increasing the exposure time of cells to 0.1 fig/ml MC from 2 h to 8 h, there was a large increase in the percentage of cells in the GJM phase as well as an increase in the cell surface area. This increase was accompanied by a significant enhancement of the cellular uptake of PII, as seen by a marked shifting of the PII fluorescence histograms to the right. PI1 alone, at the concentrations used here, had no effect on the cell cycle. Cells incubated with both MC and PI1 exhibited a 1.3-3.5-fold increase in the number of G,JM phase cells, a 1.3 -2.3-fold increase in cell surface area and a 1.3 - 2.7-fold increase in the cellular fluorescence of PI1 as compared to control cells incubated with only PII (Table I). The effects of MC on cellular PI1 uptake changed with the time interval between the removal of MC and the incubation with PI1 (Fig. 4). The increase of the fluorescence of PI1 was larger when PI1 was given at 20 h after a 4-h incubation with 0.1 pg/ml MC than when PI1 was added at 44 h or 68 h after such incubation with MC. With increasing time after exposure to MC, the changes in DNA content, cell surface area and PI1 fluorescence can recover to the level of cells treated with PI1 only. In Fig. 4, MC clearly induced an accumulation of the cells in GJM phase. The fraction of cells in GJM phase (about 55%) 20 h after incubation with MC is apparently larger than the number of cells killed by MC (20%) (Fig. 1). This indicates that a large fraction of the cells is only transiently blocked in GJM phase and will divide at a later point. Hence, it appears that most of the affected cells were recovering after exposure to MC rather than being replaced by normal cells. In order to determine the effect of dead cells on the experimental data, after the treatments of MC and PII mentioned above, the cells were incubated with propidium iodide, which is staining only for dead cells, before FCM measurement. In each sample a small proportion of dead cells (lo- 15%) was observed. The PI1 fluorescence of the dead cells was
158
DNA Histogram
CeII Surface Area PU Fluorescence
Pll only
FL1
2h MC,Pll
K
4 h MC, PI1
FL1
8h MC,Pll
l-/blLA
Fig. 3. PI1 (FL2), DNA (FLl) and cell surface area (LSl) histograms of cells which were exposed to 0.1 pg/ml MC for 2 h, 4 h and 8 h, then washed and incubated further in pure medium with 10%. FCS for 22 h, 20 h and 16 h, respectively, before they were incubated with PI1 (10 pg/ml) for 16 h. Abscissa unit is channel number (relative linear scale) representing, respectively, the intensity of fluorescence of the DNA specific dye HO 33258 (DNA histograms, FLl); the size of cells (histograms of cell surface area, LSl); the intensity of intracellular PII fluorescence (histograms of PII fluorescence, FL2). All ordinates represent the number of cells per channel (relative linear scale).
somewhat larger than that of vital cells, but this difference was not sufficient to explain the increase of the PII fluorescence of the total samples. Figure 5 shows an approximately linear rela-
tionship between the fluorescence of PII and the ceil surface area as measured by light scattering. The slope of the curves for MC-treated ce!ls was somewhat larger than for the control, which is to say that the PI1 fluorescence of cells
159
Table I. The effects of mitomycin cellular fluorescence of PII. Sample
PII (control) 2 h MC, 22 4 h MC, 20 8 h MC, 16 4 h MC, 44
h h h h
‘PM, PM, PM, PM,
16 h PII 16 h PII 16 h PI1 161 h PI1
4 h MC, 68 h PM, 16 h PI1
C on the percentage
of cells in S and Gs/M phase,
cell surface area and average
% Cells in S and GJM
Cell surface area (rel. units)
PII fluorescence (rel. units)
30 36 27 24 43
19 33 56 66 25
1 1.3 1.6 2.3 1.3
37
17
1.1
1 1.3 1.8 2.7 1.7 1.1
The cells were incubated with 0.1 rg/ml MC for 2 h, 4 h and 8 h, respectively. PI1 (10 pg/ml) was given for 16 h starting 16 - 68 h after removal of MC. Each value is from median of 8000 cells analyzed by fIow cytometry. ‘PM: pure medium with 10% FCS.
DNA Histogram
Ceil Surface Area Pll fluorescence
FL1
PI1 only
‘IL I !&LQ_ 4 I i
IS1
FL1
4h MC,20h PM*,Pll
FL1
4 h MC, 44 h PM, F’ll
FLI
4h MC,68h PM,Pll
J,
,h__
Fig. 4. Histograms of PI1 fluorescence (FL2), DNA content (FLl) and surface area (LSl) of cells which were incubated with 10 fig/ml PI1 for 16 h starting at 20 h, 44 h and 68 h, respectively, after removal of 0.1 rg/ml MC to which the cells were exposed for 4, h. The units of ordinate and abscissa are same as in Fig. 3. * PM, pure medium with 10% FCS.
160
250
E
200
6 0 z
150
6 3 z
100
a 50
,L
0
0
I
1
60
120
Cel
I
.
Surface
.
I
I
160
240
Area
Fig. 5. Cellular PI1 fluorescence as a function of cell surface area (LSl). Symbols: 0 control cells incubated with PII for 16 h; x cells were exposed to MC for 2 h. washed and incubated further in pure medium with 10% FCS for 22 h and then PII was given for 16 h; 0 cells exposed to MC for 4 h, washed and incubated in pure medium with 10% FCS for 20 h and then PI1 was added for 16 h; A 8 h, MC, washing and 16 h, pure medium with 10% FCS and then PI1 16 h; + 4 h, MC, washing and 44 h, pure medium with 10% FCS, followed by PI1 16 h; 0 4 h, MC, washing and 68 h, pure medium with 10% FCS and then PII 16 h. Each point represents approximately 500 - 1000 cells. MC concentration: 0.1 j.kg/ml, PII concentration: 10 pg/ml.
exposed to MC was higher than that of control cells of equal size. Thus, it seems that the effect of MC on PI1 uptake was related not only to cell surface area, but also to other factors, such as the permeability of the cell membrane (see Discussion).
Discussion Mitomycin C is an antitumor antibiotic with a bifunctional alkylating action. It has been used clinically for treating gastrointestinal and
other cancers [ 121. Its mode of action is believed to be mainly the formation of cross-links of DNA, retardation of the cell cycle and inhibition of cell growth [ 131. The cytotoxicity of MC is dependent on drug concentration and exposure time (Ma et al, unpublished data). The concentration applied in the present work is clinically relevant since Shimizu et al. [15] found an MC concentration of 0.1 pg/ml in plasma 1 h after a bronchial artery infusion of 20 mg MC in cancer patients. The fluorescence of PI1 in the WiDr ceils increased with increasing exposure time to MC implying that MC facilitates the cellular uptake of PII. Possible mechanisms of the effect of MC on the cellular uptake of PI1 are considered below. Cell surface area is a major determinant of the cellular uptake of PI1 (Figs. 2 and 5). Our data demonstrate that the surface area of WiDr cells increases throughout all parts of interphase (Fig. 2). The early study of Steen et al. [16] showed that the size of synchronized NHIK 3025 cells increased approximately linearly with time through the cell cycle. MC can enhance the cell surface area by retarding the cells in Gz/M phase (Fig. 3). A nearly linear correlation between the fluorescence intensity of intracellular PI1 and the cell surface area was found in Fig. 5. This finding is in agreement with the report of Christensen et al., who found that the amount of cell bound hematoporphyrin increased as cells proceeded through the cell cycle and was approximately doubled from Gl to late G2 [17]. The mechanisms by which PI1 is taken up by cells are not well known. PII, which consists primarily of lipophilic components, localizes mainly in cell membrane structures [18]. If the first step of passage into the cell is that the dye is passively dissolved in the cell membrane, the observed proportionality between the cellular uptake of PI1 and the cell surface area is to be expected. It has been suggested that LDL receptors on the cell membrane may play a role in cellular uptake of PI1 [19]. A significant fraction of PI1 binds to LDL in serum [20]. Again, it seems likely that the number of such
161
receptors is proportional to the cell surface area. Bare1 et al. [21] proposed that there may be a lower number of LDL receptors in small cells. The present finding that small cells take up relatively little of PI1 is consistent with the work of West et al. [22]. These authors reported that with increasing spheroid size of WiDr cells, there was a decrease in intracellular HPD levels, because the larger spheroids have an increased proportion of Gl-like cells with smaller volume [23]. However, it appears that cell surface area is not the only determinant of the effect of MC on the cellular uptake of PI1 (Fig. 5). Other factors, like membrane permeability may be of importance. MC has been shown to be capable of generating oxygen free radicals which produce substantial (damage to biologic membranes [24]. Therefore, one may speculate that the permeability of the cell membrane is increased probably by MC. Thus, a passive drug entry of PII into the cell (independent of a facilitated transportation by LDL receptors) may be enhanced by a toxic effect of MC on the cell membrane. Further experiments are needed to elucidate the nature of possible interactions between MC and the cell membrane. It is not clear whether the increase of intracellular PI1 induced by the effects of MC on the cell cycle is specific for tumor cells. However, the fact that tumor tissues of patients with gastric and colorectal cancers are more sensitive to MC than are adjacent normal tissues has been demonstrated [25]. They proposed that since the DNA synthesis and replication are more active in tumor tissues than in normal tissues, tumor tissues are more likely to be affected by a DNA-interacting drug such as MC. It remains to be seen whether MC enhances the uptake of PII in tumors with some selectivity.
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