Int. J . Cancer: 49, 73-76 (1991) 0 1991 Wiley-Liss, Inc.
Publication of the International Union Against Cancer Publication de I'Union Internationale Contre 18 Cancer
EPIDERMAL GROWTH FACTOR REDUCES RESISTANCE TO DOXORUBICIN Tim T. KWOKand Robert M. SUTHERLAND Laboratory of Cell and Molecular Biology, Life Sciences Division, SRI International, Menlo Park, CA 94025, USA. Epidermal growth factor (EGF) increased the sensitivity to doxorubicin (DOX) of a human squamous carcinoma cell line, A431. The relative enhancement of sensitivity by EGF was greater in 2 DOX-resistant sublines, A43 IIAS and A43 IIAIO, established by growing cells from surviving colonies after treatment of A43 I cells with DOX. A greater number of EGF receptors (both high- and low-affinity binding sites) was found for resistant A43I/AS and A43IlAIO cells than for parental cells. The enhanced drug responsiveness is not directly related to EGF effects on growth, as growth inhibition by EGF appears to be similar among the 3 sublines.
Multi-drug resistance (MDR) is one of the major obstacles encountered during chemotherapy of cancer. Tumor cells initially sensitive to a chemotherapeutic drug may develop resistance to that drug and often become resistant to several other chemotherapeutic drugs as well (Curt et a l . , 1984; Ling et a l . , 1984). An analogous situation in vitro is the development of MDR cells after culture of cells in the presence of a cytotoxic drug (Ling et a l . , 1983; van der Bliek and Borst, 1989). Cells which become resistant to one drug are normally cross-resistant to other drugs. There are several possible mechanisms for MDR. Over-expression of a transmembrane protein, P-glycoprotein, results in increase of drug efflux and reduction in drug accumulation (Beck, 1987; Moscow and Cowan, 1988; van der Bliek and Borst, 1989). Elevated activity of g1utathione-Stransferase and protein kinase C or a reduction in topoisomerase I1 activity have also been reported (Sigfried et a l . , 1983; Beck, 1987; Fine e f a / . , 1988; Kramer et al., 1988; Moscow and Cowan, 1988; Deffie et at., 1989). Some agents reported to overcome MDR are trifluoroperazine, a calmodulin antagonist; verapamil, a calcium channel blocker; cyclosporin A, an immunosuppressive agent; chloroquine, a lysosomotropic agent; monensin, a secretory inhibitor; and Tween 80, a membrane detergent (Tsuruo et al., 1982, 1984; Tsuruo, 1983; Beck et al., 1986; Slater et a l . , 1986; Twentyman et a l . , 1987; Klohs and Steinkampf, 1988; van der Bliek et al., 1989). Rodent and human cells that have developed resistance to actinomycin D or vincristine have more epidermal growth factor receptors (EGFR) than the parental line (Meyers et al., 1986, 1988). Previously, we showed that EGF enhanced cellular radiosensitivity of several human squamous-cell carcinoma (SCC) cell lines grown as monolayer cultures or small spheroids (Kwok and Sutherland, 1989, 1991). The goal of the present study was to investigate the possible interactions between EGF and DOX using an in vitro system. MATERIAL AND METHODS
The cell line used in the study is A431, a human SCC line. The line is a subclone of A431 cells from the ATCC (Rockville, MD) and differs from the ATCC line in growth rate and differentiation (Knuechel e f al., 1990). Cells were grown in Dulbecco's minimum essential medium supplemented with 10% fetal bovine serum. Development of drug-resistant lines Exponential phase culture was obtained by incubating 5 x lo5 cells in a 75-cm2 culture flask for 2 days. Cells were then exposed to 5 p,g/ml doxorubicin (DOX) for 1 hr. After being washed twice with fresh medium and trypsinized, lo6 cells were transferred to a 75-cm2 culture flask. Cells were then
allowed to grow under drug-free conditions, with medium change twice a week, until they reached 80% confluence (about 3 months). Thereafter, cells were trypsinized and passaged twice and labelled as A431lA5. The subline A43llA5 was used in another similar selection process in which the dose of DOX was increased from 5 to 10 p,g/ml. The subline developed was called A43 1/A 10. Protocol of experiment Exponential phase cells were used in this study. The cultures were obtained by growing 2.5 X lo5 cells in 5 ml of medium in a 60-mm Petri dish for 2 days. After being exposed to various concentrations of DOX for 1 hr, the cells were rinsed twice with fresh medium. After trypsinization, cell survival was measured by clonogenic assay, as follows. Various dilutions of cells were aliquoted into 60-mm Petri dishes and allowed to grow for 12 days. At the end of the incubation, the dishes were stained by methylene blue, and colonies of more than 50 cells were scored. Murine EGF (Collaborative Research, Bedford, MA) of 50 ng/ml was present throughout the experiments ( i . e . , the 2-day growth period, the time of drug exposure, and the time of colony formation). Results from preliminary experiments indicated that 50 ng/ml of EGF maximized the sensitization of DOX response in the parental cells. EGF present before and during or after the treatment with DOX demonstrated a sensitization effect whereas continuous exposure, i . e . a combination of both, appeared to have the greatest effect. Cell growth studies After exposure to DOX as described above, lo5 cells in 5 ml of medium were aliquoted into 60-mm Petri dishes. EGF of 50 ng/ml was present before, during and after the drug treatment. After day 3, the medium and EGF were changed every other day. The number of cells in each plate was counted with a hemocytometer .
EGF receptor measurement Cells (2 x lo5) in 2 ml of medium were grown in one well of a 6-well plate for 2 days. After being washed twice with serum-free medium supplemented with 1% bovine serum albumin (BSA-medium), the cells were incubated with various amounts of 1251-EGFfor 4 hr at 4°C. At the end of incubation, the cells were washed 3 times with BSA-medium and lysed in 1 ml of IM NaOH and 0.5% SDS solution. The radioactivity was counted in a gamma counter. Non-specific binding was assessed by incubating cells with 1 mM cold EGF. The data were analyzed by Scatchard analysis. RESULTS
The dose-response curves to DOX of different sublines of A431 cells are shown in Figure 1. Both A431/A5 and A431/ A10 cells were more resistant to DOX than their parental line
'To whom correspondence and reprint requests should be sent, at the Laboratory of Cell and Molecular Biology, SRI International, 333 Ravenswood Ave., Menlo Park, CA 94025. Received: December 1 I , 1990 and in revised form April 15, 1991.
74
KWOK AND SUTHERLAND
C 0 N C E NTR ATlON (yg/ml) C O N C E N T R A T I O N (pg/rni)
FIGUREi - Dose-response curve to DOX of A431/p A43i/A5 cells (,-J), Results are averaged from separate experiments. Error bars: SD.
(o),
(A,and A431/A]0
FIGURE 2 - Dose-response curve to DOX of (a)A43 Up, (b) A43 1/ A5 and (c) A431iA10 cells. Open symbols: untreated control cultures. were exposed to So ng/ml EGF throughout the experiment (see ‘‘Material and Methods”). Results are averaged from 3 separate experiments. Error bars: SD.
TABLE I - CELL GROWTH PARAMETERS Sublines
% Plating efficiency
DOX‘
No EGF
A431/p
-
84
f
lo3
Doubling time (hr) Na EGF
With EGF~
No EGF
With EGF
36 f 5
26 f 5 43 t 10 2526 65 t 14 24 t 8 62 t 13
22 t 4 38 2 9 2124 64 2 11 22 t 5 60 ? 10
5?1 34 f 8 10?1 25 f 8 10 k 2 34 Sr 9
17t2 67 t 14 29t4 58 t 11 31 t 6 72 C 14
i-
A431/A5 A431/A10
-
+ +-
74218
3026
54
12f 5
f
10
Lag phase (hr)
With EGF
‘Cells were exposed to I pgiml DOX for I hr.-’EGF of 50 ngiml was present during the 2-day growth period and throughout the course of growth ~tudies.-~Mean2 SD.
(A4311p cells). Continuous exposure to 50 ngiml EGF ( i . e . , during the 2-day growth period, the time of drug exposure, and the 12-day colony formation period) increased the sensitivity to DOX of the sublines of A431 cells (Fig. 2). Similar results were demonstrated if the clonogenic assay period was extended from 12 to 19 days (results not shown). The ratio between the surviving fraction (at 2.5 pgiml DOX) of untreated controls and EGF-treated cells is 20.0 for A431lA10, 5.8 for A431lA5, and 3.3 for A43I/p cells (Fig. 2). The continuous exposure to EGF in these experiments reduced the plating efficiency (PE) of all 3 sublines by about 60% to 80% of the values for untreated controls (Table I). The doubling time (DT) for all 3 control cultures was almost the same, about 24 hr, whereas about a 2-fold increase in lag phase was seen for the 2 DOX-resistant sublines (Table I). Treatment with DOX increased both the DT and the lag phase of all 3 sublines with or without continuous exposure to EGF. Although the DT for the 2 resistant lines after treatment with DOX is slightly longer than for the parental cells, the difference is not statistically significant. In all untreated control and DOX-treated cells, EGF did not affect the DT but increased by about 2 to 3 times the lag period of cell growth (Table I). The biphasic Scatchard plot for the binding of EGF to its receptor indicates that there are high- and low-affinity binding sites in all 3 sublines (Fig. 3). The 2 resistant sublines had a higher total receptor number than A431/p cells, associated with increases in both high- and low-affinity binding sites (Table 11). The binding affinities for the high- and low-affinity sites were similar among the 3 sublines (Table 11).
B (prnole/lo6cells) FIGURE 3 - Scatchard plot for EGF receptor binding of A431/p
(O), A431iA5 (A), and A431/A10 cells (a).B: bound EGF; F: free EGF. Results are from one experiment.
DISCUSSION
Epidermal growth factor sensitizes the response of cells to DOX and the relative effect is greater for the drug-resistant cell lines. Drug sensitization is also observed for transforming growth factor a,a EGF-like growth factor (results not shown). Epidermal growth factor may overcome drug resistance through at least 2 different general mechanisms. Cells showing DOX resistance may over-express P-glycoprotein, glutathioneS-transferase, protein kinase C or have a reduction in topo-
75
EGF AND DOX RESISTANCE TABLE I1 - AFFINITY AND NUMBER OF EGFR IN DIFFERENT SUBLINES High-affinity
A43 Up A431/A5 A431iA10 'Mean
0.13 + 0.02' 0.16 + 0.04 0.20 k 0.06
Low-affini~y
1.34 2 0.05 2.36 0.28 3.36 0.66
*
*
3.76 2 0.67 3.09 t 0.23 3.38 2 0.49
1.42 2.39 2.39
* 0.14 + 0.20 * 0.45
1.55 t 0.14 2.60 ? 0.20 2.73 t 0.45
f SD.
isomerase I1 activity (Sigfried et al., 1983; Beck, 1987; Fine et al., 1988; Kramer etal., 1988; Moscow and Cowan, 1988; van der Bliek et al., 1989; Deffie et al., 1989). The signal transduction process initiated by EGF involves binding of the growth factor to its receptor, activation of various kinds of kinase (such as tyrosine kinase, protein kinase C), and phospholipase C. Subsequent changes include alterations in cellular polyphosphoinositols, calcium levels, and the induction of c-fos (Carpenter, 1987). It is possible that the EGF signal transduction process may interact with the drug-resistance mechanism(s) in some common metabolic pathways and thereby result in sensitization. Another possible interaction between EGF and resistance mechanisms is the turnover pathway of the plasma membrane. Often drug resistance is associated with reduced accumulation of drug by increased efflux. Retention of DOX was increased in the presence of lysosomotrophic drugs or a secretory inhibitor which affects the process of endocytosis (Beck, 1987; Klohs and Steinkampf, 1988). The drug is retained within the lysosomes and, later on, is either transported to the active target or excluded by fusion of the Iysosome with plasma membrane (Beck, 1987; Klohs and Steinkampf, 1988). EGF binding to EGFR results in internalization of the receptor complex involving endocytosis. This could result in more DOX uptake if the drug bound to the cell membrane. The growth factor is degraded within the lysosome and later excreted (Carpenter, 1987; Sorkin et al., 1989). In the presence of growth factor, the cells may be overwhelmed by the membrane-recycling process induced by EGF. This in turn could reduce their capability to excrete the drug, and increases the probability of having the drug delivered to the target site.
Increases in EGFR density are reported in rodent and human cell lines which exhibit drug resistance (Meyers et al., 1986, 1988). In general, the EGFR affinity of the sensitive and resistant cells does not differ. Our results agree with all but the data from the large-cell lung carcinoma which demonstrated reduction in EGFR as a result of loss of the high-affinity binding site (Reeve et a l . , 1990). The correlation of EGFR density and drug resistance is not yet clear, although DOX was shown to upregulate EGFR in cells (Zuckier and Tritton, 1983). The genes for EGFR and P-glycoproteins in human cells are located on chromosome 7, but the expression of these 2 genes is not always directly correlated (Meyers et al., 1988). Our previous results demonstrate that EGF enhances the radiosensitivity of several human SCC lines which express high levels of EGF receptor but not those with low receptor number (Kwok and Sutherland, 1991). This may also be the case for EGF-induced DOX sensitization. No sensitization by EGF of the response to DOX was seen in another SCC line, SiHa cells, which expressed only about lo5 EGF receptors/cell (results not shown). Co-administration of EGF with 5-fluorouracil and cisplatin extended the time for growth delay of tumor in uivo (Amagase et al., 1989). Further characterization of the EGF effect in the present in uitro system on responses to other anti-cancer drugs, such as cisplatin and 5-fluorouracil, may help in understanding the basis of the effects of EGF in drug and radiation sensitivity and the possible use of this and other growth factors in treatment of cancer. ACKNOWLEDGEMENTS
This work was supported by NIH grant CA-37618.
REFERENCES AMAGASE, H., KAKIMOTO, M., HASHIMOTO, K., FUWA,T. and TSUKAGOSHI,S., Epidermal growth factor receptor-mediated selective cytotoxicity of antitumor agents toward human xenografts and murine syngeneic solid tumors. Jap. J . Cancer Res., 80, 670-678 (1989). BECK,W.T., The cell biology of multiple drug resistance. Biochem. Pharmacol., 36, 2879-2887 (1987). BECK,W.T., CIRTAIN,M.C., LOOK,A.T. and ASHMUN, R.A., Reversal of Vinca alkaloid resistance but not multiple drug resistance in human leukemic cells by verapamil. Cancer Res., 46, 778-784 (1986). CARPENTER, G., Receptors for epidermal growth factor and other polypeptide mitogens. Ann. Rev. Biochem., 56, 881-914 (1987). CURT,G.A., CLENDENINN, N.J. and CHABNER, B.A., Drug resistance in cancer. Cancer Treat. Rep., 68, 87-99 (1984). DEFFIE,A.M., BOSMAN,D.J. and GOLDENBERG, G.J., Evidence for a mutant allele of the gene for DNA topoisomerase I1 in adriamycin-resistant P388 murine leukemia cells. Cancer Res., 49, 6879-6882 (1989). FINE,R.L., PATEL,J. and CHABNER, B.A., Phorbol esters induce multidrug resistance in human breast cancer cells. Proc. nut. Acad. Sci. (Wash.), 85, 582-586 (1988). KLOHS,W.D. and STEINKAMPF, R.W., The effect of lysosomotropic agents and secretoty inhibitors on anthracycline retention and activity in multiple drug resistant cells. Mol. Pharmacol., 34, 180-185 (1988). KNUECHEL, R., KING, P . , HOFSTOEDTER, J., LANGMUIR, V., SUTHER-
LAND,R.M. and PENNY,D.P., Differentiation patterns in two- and threedimensional culture systems of human squamous carcinoma cell lines. Amer. J . Pathol., 137, 725-736 (1990). KRAMER, R.A., ZAKHER,J. and KIM, G., Role of the glutathione redox cycle in acquired and de now multidrug resistance. Science, 241,694-697 (1988). KWOK,T.T. and SUTHERLAND, R.M., Enhancement of sensitivity of human squamous carcinoma cells to radiation by epidermal growth factor. J . nat. Cancer Inst., 81, 1020-1024 (1989). KWOK,T.T. and SUTHERLAND, R.M., Differences in EGF related radiosensitization of cells with high and low numbers of EGF receptors. Brit. J . Cancer, in press. LING, V., GERLACH, J. and KARTNER, N . , Multidrug resistance. Breast Cancer Res. Treat., 4, 89-94 (1984). LING,V., KARTNER, N., SUDO,T., SIMINOVITCH, L. and RIORDAN, J.R., Multidrug-resistance phenotype in Chinese hamster ovary cells. Cancer Treat. Rep., 67, 869-874 (1983). MEYERS,M.B., MERLUZZI,V.J., SPENGLER,B.A. and BIEDLER,J.L., Epidermal growth factor receptor is increased in multidrug resistant Chinese hamster and mouse tumor cells. Proc. nat. Acad. Sci. (Wash.), 83, 5521-5525 (1986). MEYERS,M.B., VIOLETSHEN.W.P., SPENGLER, B.A., CICCARONE, V., O'BRIEN,J.P., DONNER,D.B., FURTH,M.E. and BIEDLER,J.L., In-
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KWOK AND SUTHERLAND
creased epidermal growth factor receptor in multidrug-resistant human neuroblastoma cells. J . cell. Eiochem., 38, 87-97 (1988). Moscow, J.A. and COWAN,K.H., Multidrug resistance. J. naf. Cancer Znsr., 80, 14-20 (1988). REEVE,J.G., RABBITTS,P.H. and TWENTYMAN, P.R., Non-P-glycoprotein-mediated multidrug resistance with reduced EGF receptor expression in a human large cell lung cancer cell line. Erif. J . Cuncer, 61, 851-854 (1990). SIGFRIED,J., TRITTON,T.R. and SARTORELLI, A.C., Comparison of anthracycline concentrations in S180 cell lines of varying sensitivity. Europ. J. Cancer d i n . Oncol., 19, 1133-1141 (1983).
SLATER,L., SWEET,P., STUPECKY, M., WETZEL,M. and GUPTA,S., Cyclosporin A corrects daunorubicin resistance in Ehrlich ascites carcinoma. Brit. J. Cancer, 54, 235-238 (1986). SORKIN,A., KORNILOVA, E., TESLENKO, L., SOROKIN, A. and NIKOLSKY, N., Recycling of epidermal growth factor-receptor complexes in A431 cells. Biochim. Eiophys. Acra, 1011, 88-96 (1989). TSURUO,T., Reversal of acquired resistance to Vinca alkaloids and anthracycline antibiotics. Cancer Treat. Rep., 67, 889-894 (1983).
TSURUO,T., IIDA,H., KITATANI,Y., YOKOTA,K., TSUKAGOSHI, S. and Y., Effects of quinidine and related compounds on cytotoxicity: SAKURAI, and cellular accumulation of vincristine and adriamycin in drug-resistant tumor cells. Cancer Res., 44, 43034307 (1984). TSURUO,T., IIDA,H., TSUKAGOSHI, S. and SAKURAI, Y., Increased accumulation of vincristine and adriamycin in drug-resistant P388 tumor cells following incubation with calcium antagonists and calmodulin inhibitors. Cuncer Res., 42, 473W733 (1982). TWENTYMAN, P.R., Fox, N. and WHITE,D., Cyclosporin A and its analogues as modifiers of adriamycin and vincristine resistance in multi-drug resistant human lung cancer cell lines. Brit. J. Cancer, 56, 55-57 (1987). VAN DER BLIEK,A.M. and BORST,P., Multidrug resistance. Advanc. Cancer Res., 52, 165-203 (1989).
ZUCKIER,G. and TRITTON,T.R., Adriamycin causes up regulation of epidermal growth factor receptors in actively growing cells. Exp. Cell Res., 148, 155-161 (1983).
Publication of the International Union Against Cancer Publication de I'Union Internationale Contre 18 Cancer
EPIDERMAL GROWTH FACTOR REDUCES RESISTANCE TO DOXORUBICIN Tim T. KWOKand Robert M. SUTHERLAND Laboratory of Cell and Molecular Biology, Life Sciences Division, SRI International, Menlo Park, CA 94025, USA. Epidermal growth factor (EGF) increased the sensitivity to doxorubicin (DOX) of a human squamous carcinoma cell line, A431. The relative enhancement of sensitivity by EGF was greater in 2 DOX-resistant sublines, A43 IIAS and A43 IIAIO, established by growing cells from surviving colonies after treatment of A43 I cells with DOX. A greater number of EGF receptors (both high- and low-affinity binding sites) was found for resistant A43I/AS and A43IlAIO cells than for parental cells. The enhanced drug responsiveness is not directly related to EGF effects on growth, as growth inhibition by EGF appears to be similar among the 3 sublines.
Multi-drug resistance (MDR) is one of the major obstacles encountered during chemotherapy of cancer. Tumor cells initially sensitive to a chemotherapeutic drug may develop resistance to that drug and often become resistant to several other chemotherapeutic drugs as well (Curt et a l . , 1984; Ling et a l . , 1984). An analogous situation in vitro is the development of MDR cells after culture of cells in the presence of a cytotoxic drug (Ling et a l . , 1983; van der Bliek and Borst, 1989). Cells which become resistant to one drug are normally cross-resistant to other drugs. There are several possible mechanisms for MDR. Over-expression of a transmembrane protein, P-glycoprotein, results in increase of drug efflux and reduction in drug accumulation (Beck, 1987; Moscow and Cowan, 1988; van der Bliek and Borst, 1989). Elevated activity of g1utathione-Stransferase and protein kinase C or a reduction in topoisomerase I1 activity have also been reported (Sigfried et a l . , 1983; Beck, 1987; Fine e f a / . , 1988; Kramer et al., 1988; Moscow and Cowan, 1988; Deffie et at., 1989). Some agents reported to overcome MDR are trifluoroperazine, a calmodulin antagonist; verapamil, a calcium channel blocker; cyclosporin A, an immunosuppressive agent; chloroquine, a lysosomotropic agent; monensin, a secretory inhibitor; and Tween 80, a membrane detergent (Tsuruo et al., 1982, 1984; Tsuruo, 1983; Beck et al., 1986; Slater et a l . , 1986; Twentyman et a l . , 1987; Klohs and Steinkampf, 1988; van der Bliek et al., 1989). Rodent and human cells that have developed resistance to actinomycin D or vincristine have more epidermal growth factor receptors (EGFR) than the parental line (Meyers et al., 1986, 1988). Previously, we showed that EGF enhanced cellular radiosensitivity of several human squamous-cell carcinoma (SCC) cell lines grown as monolayer cultures or small spheroids (Kwok and Sutherland, 1989, 1991). The goal of the present study was to investigate the possible interactions between EGF and DOX using an in vitro system. MATERIAL AND METHODS
The cell line used in the study is A431, a human SCC line. The line is a subclone of A431 cells from the ATCC (Rockville, MD) and differs from the ATCC line in growth rate and differentiation (Knuechel e f al., 1990). Cells were grown in Dulbecco's minimum essential medium supplemented with 10% fetal bovine serum. Development of drug-resistant lines Exponential phase culture was obtained by incubating 5 x lo5 cells in a 75-cm2 culture flask for 2 days. Cells were then exposed to 5 p,g/ml doxorubicin (DOX) for 1 hr. After being washed twice with fresh medium and trypsinized, lo6 cells were transferred to a 75-cm2 culture flask. Cells were then
allowed to grow under drug-free conditions, with medium change twice a week, until they reached 80% confluence (about 3 months). Thereafter, cells were trypsinized and passaged twice and labelled as A431lA5. The subline A43llA5 was used in another similar selection process in which the dose of DOX was increased from 5 to 10 p,g/ml. The subline developed was called A43 1/A 10. Protocol of experiment Exponential phase cells were used in this study. The cultures were obtained by growing 2.5 X lo5 cells in 5 ml of medium in a 60-mm Petri dish for 2 days. After being exposed to various concentrations of DOX for 1 hr, the cells were rinsed twice with fresh medium. After trypsinization, cell survival was measured by clonogenic assay, as follows. Various dilutions of cells were aliquoted into 60-mm Petri dishes and allowed to grow for 12 days. At the end of the incubation, the dishes were stained by methylene blue, and colonies of more than 50 cells were scored. Murine EGF (Collaborative Research, Bedford, MA) of 50 ng/ml was present throughout the experiments ( i . e . , the 2-day growth period, the time of drug exposure, and the time of colony formation). Results from preliminary experiments indicated that 50 ng/ml of EGF maximized the sensitization of DOX response in the parental cells. EGF present before and during or after the treatment with DOX demonstrated a sensitization effect whereas continuous exposure, i . e . a combination of both, appeared to have the greatest effect. Cell growth studies After exposure to DOX as described above, lo5 cells in 5 ml of medium were aliquoted into 60-mm Petri dishes. EGF of 50 ng/ml was present before, during and after the drug treatment. After day 3, the medium and EGF were changed every other day. The number of cells in each plate was counted with a hemocytometer .
EGF receptor measurement Cells (2 x lo5) in 2 ml of medium were grown in one well of a 6-well plate for 2 days. After being washed twice with serum-free medium supplemented with 1% bovine serum albumin (BSA-medium), the cells were incubated with various amounts of 1251-EGFfor 4 hr at 4°C. At the end of incubation, the cells were washed 3 times with BSA-medium and lysed in 1 ml of IM NaOH and 0.5% SDS solution. The radioactivity was counted in a gamma counter. Non-specific binding was assessed by incubating cells with 1 mM cold EGF. The data were analyzed by Scatchard analysis. RESULTS
The dose-response curves to DOX of different sublines of A431 cells are shown in Figure 1. Both A431/A5 and A431/ A10 cells were more resistant to DOX than their parental line
'To whom correspondence and reprint requests should be sent, at the Laboratory of Cell and Molecular Biology, SRI International, 333 Ravenswood Ave., Menlo Park, CA 94025. Received: December 1 I , 1990 and in revised form April 15, 1991.
74
KWOK AND SUTHERLAND
C 0 N C E NTR ATlON (yg/ml) C O N C E N T R A T I O N (pg/rni)
FIGUREi - Dose-response curve to DOX of A431/p A43i/A5 cells (,-J), Results are averaged from separate experiments. Error bars: SD.
(o),
(A,and A431/A]0
FIGURE 2 - Dose-response curve to DOX of (a)A43 Up, (b) A43 1/ A5 and (c) A431iA10 cells. Open symbols: untreated control cultures. were exposed to So ng/ml EGF throughout the experiment (see ‘‘Material and Methods”). Results are averaged from 3 separate experiments. Error bars: SD.
TABLE I - CELL GROWTH PARAMETERS Sublines
% Plating efficiency
DOX‘
No EGF
A431/p
-
84
f
lo3
Doubling time (hr) Na EGF
With EGF~
No EGF
With EGF
36 f 5
26 f 5 43 t 10 2526 65 t 14 24 t 8 62 t 13
22 t 4 38 2 9 2124 64 2 11 22 t 5 60 ? 10
5?1 34 f 8 10?1 25 f 8 10 k 2 34 Sr 9
17t2 67 t 14 29t4 58 t 11 31 t 6 72 C 14
i-
A431/A5 A431/A10
-
+ +-
74218
3026
54
12f 5
f
10
Lag phase (hr)
With EGF
‘Cells were exposed to I pgiml DOX for I hr.-’EGF of 50 ngiml was present during the 2-day growth period and throughout the course of growth ~tudies.-~Mean2 SD.
(A4311p cells). Continuous exposure to 50 ngiml EGF ( i . e . , during the 2-day growth period, the time of drug exposure, and the 12-day colony formation period) increased the sensitivity to DOX of the sublines of A431 cells (Fig. 2). Similar results were demonstrated if the clonogenic assay period was extended from 12 to 19 days (results not shown). The ratio between the surviving fraction (at 2.5 pgiml DOX) of untreated controls and EGF-treated cells is 20.0 for A431lA10, 5.8 for A431lA5, and 3.3 for A43I/p cells (Fig. 2). The continuous exposure to EGF in these experiments reduced the plating efficiency (PE) of all 3 sublines by about 60% to 80% of the values for untreated controls (Table I). The doubling time (DT) for all 3 control cultures was almost the same, about 24 hr, whereas about a 2-fold increase in lag phase was seen for the 2 DOX-resistant sublines (Table I). Treatment with DOX increased both the DT and the lag phase of all 3 sublines with or without continuous exposure to EGF. Although the DT for the 2 resistant lines after treatment with DOX is slightly longer than for the parental cells, the difference is not statistically significant. In all untreated control and DOX-treated cells, EGF did not affect the DT but increased by about 2 to 3 times the lag period of cell growth (Table I). The biphasic Scatchard plot for the binding of EGF to its receptor indicates that there are high- and low-affinity binding sites in all 3 sublines (Fig. 3). The 2 resistant sublines had a higher total receptor number than A431/p cells, associated with increases in both high- and low-affinity binding sites (Table 11). The binding affinities for the high- and low-affinity sites were similar among the 3 sublines (Table 11).
B (prnole/lo6cells) FIGURE 3 - Scatchard plot for EGF receptor binding of A431/p
(O), A431iA5 (A), and A431/A10 cells (a).B: bound EGF; F: free EGF. Results are from one experiment.
DISCUSSION
Epidermal growth factor sensitizes the response of cells to DOX and the relative effect is greater for the drug-resistant cell lines. Drug sensitization is also observed for transforming growth factor a,a EGF-like growth factor (results not shown). Epidermal growth factor may overcome drug resistance through at least 2 different general mechanisms. Cells showing DOX resistance may over-express P-glycoprotein, glutathioneS-transferase, protein kinase C or have a reduction in topo-
75
EGF AND DOX RESISTANCE TABLE I1 - AFFINITY AND NUMBER OF EGFR IN DIFFERENT SUBLINES High-affinity
A43 Up A431/A5 A431iA10 'Mean
0.13 + 0.02' 0.16 + 0.04 0.20 k 0.06
Low-affini~y
1.34 2 0.05 2.36 0.28 3.36 0.66
*
*
3.76 2 0.67 3.09 t 0.23 3.38 2 0.49
1.42 2.39 2.39
* 0.14 + 0.20 * 0.45
1.55 t 0.14 2.60 ? 0.20 2.73 t 0.45
f SD.
isomerase I1 activity (Sigfried et al., 1983; Beck, 1987; Fine et al., 1988; Kramer etal., 1988; Moscow and Cowan, 1988; van der Bliek et al., 1989; Deffie et al., 1989). The signal transduction process initiated by EGF involves binding of the growth factor to its receptor, activation of various kinds of kinase (such as tyrosine kinase, protein kinase C), and phospholipase C. Subsequent changes include alterations in cellular polyphosphoinositols, calcium levels, and the induction of c-fos (Carpenter, 1987). It is possible that the EGF signal transduction process may interact with the drug-resistance mechanism(s) in some common metabolic pathways and thereby result in sensitization. Another possible interaction between EGF and resistance mechanisms is the turnover pathway of the plasma membrane. Often drug resistance is associated with reduced accumulation of drug by increased efflux. Retention of DOX was increased in the presence of lysosomotrophic drugs or a secretory inhibitor which affects the process of endocytosis (Beck, 1987; Klohs and Steinkampf, 1988). The drug is retained within the lysosomes and, later on, is either transported to the active target or excluded by fusion of the Iysosome with plasma membrane (Beck, 1987; Klohs and Steinkampf, 1988). EGF binding to EGFR results in internalization of the receptor complex involving endocytosis. This could result in more DOX uptake if the drug bound to the cell membrane. The growth factor is degraded within the lysosome and later excreted (Carpenter, 1987; Sorkin et al., 1989). In the presence of growth factor, the cells may be overwhelmed by the membrane-recycling process induced by EGF. This in turn could reduce their capability to excrete the drug, and increases the probability of having the drug delivered to the target site.
Increases in EGFR density are reported in rodent and human cell lines which exhibit drug resistance (Meyers et al., 1986, 1988). In general, the EGFR affinity of the sensitive and resistant cells does not differ. Our results agree with all but the data from the large-cell lung carcinoma which demonstrated reduction in EGFR as a result of loss of the high-affinity binding site (Reeve et a l . , 1990). The correlation of EGFR density and drug resistance is not yet clear, although DOX was shown to upregulate EGFR in cells (Zuckier and Tritton, 1983). The genes for EGFR and P-glycoproteins in human cells are located on chromosome 7, but the expression of these 2 genes is not always directly correlated (Meyers et al., 1988). Our previous results demonstrate that EGF enhances the radiosensitivity of several human SCC lines which express high levels of EGF receptor but not those with low receptor number (Kwok and Sutherland, 1991). This may also be the case for EGF-induced DOX sensitization. No sensitization by EGF of the response to DOX was seen in another SCC line, SiHa cells, which expressed only about lo5 EGF receptors/cell (results not shown). Co-administration of EGF with 5-fluorouracil and cisplatin extended the time for growth delay of tumor in uivo (Amagase et al., 1989). Further characterization of the EGF effect in the present in uitro system on responses to other anti-cancer drugs, such as cisplatin and 5-fluorouracil, may help in understanding the basis of the effects of EGF in drug and radiation sensitivity and the possible use of this and other growth factors in treatment of cancer. ACKNOWLEDGEMENTS
This work was supported by NIH grant CA-37618.
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