Vol. 84, No. 2, January 15, 1992
targets. Thus, the use of IL-2 may represent a uniquely attractive approach to enhance MAb penetration into tumor sites and render MAb therapy more effective.
(16) PEACE
DJ.
CHEEVER
MA:
Toxicity
and
therapeutic efficacy of high-dose interleukin 2. In vivo infusion of antibody to NK-1.1 attenuates toxicity without compromising efficacy against murine leukemia. J Exp Med 169:161-173.1989 (17) ORTALDO JR, MASON A, OVERTON R: Lym-
References
phokine-activated killer cells. Analysis of progenitors and effectors. J Exp Med 164:1193-1205.1986
(/) BERINSTEIN N. LEVY R: Treatment of a murine
B cell lymphoma with monoclonal antibodies and IL-2. J Immunol 139:971-976. 1987 (2) BERINSTEIN N. STARNES CO. LEVY R: Specific
enhancement of the therapeutic effect of antiidiotype antibodies on a murine B cell lymphoma by IL-2. J Immunol 140:2839-2845. 1988 (3) SCHULTZ KR. KLARNET JP, PEACE DJ. ET AL:
Monoclonal antibody therapy of murine lymphoma: Enhanced efficacy by concurrent administration of interleukin 2 or lymphokineactivated killer cells. Cancer Res 50:5421 — 5425. 1990
Determinants of Response to the DNA Topoisomerase II Inhibitors Doxorubicin and Etoposide in Human Lung Cancer Cell Lines
(4) PRESS OW, EARY JF, BADGER CC. ET AL:
Treatment of refractory non-Hodgkins lymphoma with radiolabeled MB-1 (anti-CD37) antibody. J Clin Oncol 7:1027-1038. 1989 (5) ROSENSTEIN M, ETTINCHAUSEN SE. ROSENBERG
SA: Extravasation of intravascular fluid mediated by the systemic administration of recombinant interleukin-2. J Immunol 137:1735-1742,1986 (6) DAMLE NK. DOYLE LV. BENDER JR. ET AL: In-
Kazuo Kasahara, Yasuhiro Fujiwara, Yoshikazu Sugimoto, Kazuto Nishio, Tomohide Tamura, Tamotsu Matsuda, Nagahiro Saijo*
terleukin 2-activated human lymphocytes exhibit enhanced adhesion to normal vascular endothelial cells and cause their lysis. J Immunol 138:1779-1785, 1987 (7) SCULIER JP, BODY JJ, NEJAI S, ET AL: En-
dogenous production of tumor necrosis factoralpha in cancer patients during adoptive immunotherapy with interleukin-2 (IL-2). Proc AACR 29:4093, 1988 (8) LOTZE MT. MATORY YL. ETTINGHAUSEN SE.
ET AL: In vivo administration of purified human interleukin 2. II. Half life, immunologic effects, and expansion of peripheral lymphoid cells in vivo with recombinant IL-2. J Immunol 135:2865-2876, 1985 (9) BADGER CC, KROHN KA, PETERSON AV, ET
AL: Experimental radiotherapy of murine lymphoma with l3lI-labeled anti-Thy I.I monoclonal antibody. Cancer Res 45:1536-1544, 1985 (10) NOWINSKI RC, HAYS EF, DOYLE T, ET AL: On-
comaviruses produced by murine leukemia cells in culture. Virology 81:363-370, 1977 (//)
DENKERS EY, BADGER CC, LEDBETTER JA, ET
AL: Influence of antibody isotype on the passive serotherapy of lymphoma. J Immunol 135:2183-2186,1985 (12) BRADFORD MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254, 1976 (13) COLTON T, ED: Statistics in Medicine. Boston: Little, Brown, 1974 (14) ROSENBERG SA, LOTZE MT, MUUL LM, ET AL:
Observations on the systemic administration of autologous lymphokine-activated killer cells and recombinant interleukin-2 to patients with metastatic cancer. N Engl J Med 313:1485— 1492,1985 (15) GRIMM EA, MAZUMDER A, ZHANG HS, ET AL:
Lymphokine-activated killer cell phenomenon. Lysis of natural killer-resistant fresh solid tumor cells by interleukin 2-activated autologous human peripheral blood lymphocytes. J Exp Med 155:1823-1841, 1982
Background: Small-cell lung cancer (SCLC) is more sensitive to anticancer agents than non-small-cell lung cancer (NSCLC), but few studies have analyzed the mechanisms of natural drug resistance responsible for this difference. Purpose: To elucidate these mechanisms, we determined drug sensitivity and evaluated the biochemical parameters affecting response to the DNA topoisomerase II inhibitors doxorubicin and etoposide in both types of cancer cell lines, in particular the activity and content of DNA topoisomerase II, as well as etoposide uptake and cell doubling time. Methods: Drug sensitivity and cellular uptake of etoposide were determined by clonogenic assay and accumulation of radiolabeled drug, respectively. The topoisomerase II activity was assayed by decatenation of kinetoplast DNA to minicircle DNA using nuclear protein, and the content was determined by immunoblot analysis of nuclear extracts. We also compared the topoisomerase II content in parent cell lines with that in lines with cisplatin resistance acquired in vitro. Results: Sensitivities to doxorubicin and etoposide were higher in SCLC cell lines than in NSCLC lines, and the REPORTS 113
Downloaded from http://jnci.oxfordjournals.org/ at University of Birmingham on August 27, 2014
cells in vitro (6) and may cause endothelial damage in vivo resulting in extravasation of intravascular fluid. TNF-a and IFN-y (14,15), released by LAK cells, may also contribute to increased capillary permeability. The effects of IL-2 in tumors are even more complex, since augmentation of the Fc receptor-bearing LAK cell population with the natural killer phenotype has been shown to be the main population of LAK cells causing capillary leak in vivo (16,17). This population can lyse tumor tissues through the mechanism of antibody-dependent cellular cytotoxicity, which may affect subsequent antibody uptake in tumors. IL-2-induced LAK cells may also lyse tumor cells, leading to release of Thy 1 antigen and formation of antibody-antigen complexes. Either of these two mechanisms may result in decreased accumulation of MAb at the tumor site. The efficacy of combined IL-2 and MAb therapy might be enhanced by changing the MAb-dosing schedule in relationship to IL-2 administration, since this maneuver could potentially increase the localization of MAb at the tumor site by taking advantage of a time period of greater capillary permeability induced by high-dose IL-2. Additionally, our data demonstrated similar biodistribution of MAb and albumin in IL-2-treated hosts (Figs. 1, 2). Consequently, approaches using smaller molecules, such as F(ab')2 fragments, could result in more selective extravasation of the MAb without the extracellular edema associated with the present regimen. Our results strongly suggest that IL-2 therapy can enhance the delivery of a radioisotope to a tumor site via a tumorspecific MAb. Moreover, since high doses of IL-2 cause activation of in vivo LAK effector cells, which are able to mediate antibody-dependent cellular cytotoxicity in vitro (1-3), the combination of IL-2 and MAb might also prove beneficial for therapy using nonradiolabeled MAb. Using lower nontoxic doses of IL-2 in combination with 1A14 MAb, we (3) have previously reported augmented antibody-induced regression of tumors. The use of higher doses of IL-2 may not only induce Fc receptor-positive antibody-dependent cellular cytotoxicity LAK effector cells, but may also result in increased amounts of MAb on the tumor
Lung cancer consists of two phenotypically distinct forms: small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC). One distinctive difference between SCLC and NSCLC is chemosensitivity. SCLC is more sensitive
Received May 23, 1991; revised November 7, 1991; accepted November 13, 1991. Supported by Grants-in-Aid from the Ministry of Health and Welfare, Japan, for the Comprehensive 10-Year Strategy for Cancer Control. K. Kasahara, Pharmacology Division. National Cancer Center Research Institute, Tokyo. Japan, and Department of Third Internal Medicine. Kanazawa University, School of Medicine, Kanazawa, Japan. Y. Fujiwara, Y. Sugimoto, K. Nishio, N. Saijo. Pharmacology Division, National Cancer Center Research Institute. T. Tamura, Department of Internal Medicine. National Cancer Center Hospital, Tokyo. T. Matsuda, Department of Third Internal Medicine, Kanazawa University, School of Medicine. We thank John Stephan Lazo. Department of Pharmacology. Pittsburgh University, for his critical review of the manuscript. *Correspondence to: Nagahiro Saijo. M.D.. Pharmacology Division, National Cancer Center Research Institute. Tsukiji 5-1-1, Chuo-ku. Tokyo 104, Japan.
114
to anticancer agents than NSCLC (/). Nakagawa et al. (2) reported that expression of glutathione S-transferase n messenger RNA in three SCLC cell lines and three NSCLC cell lines inversely correlated with sensitivity to cisplatin and carboplatin, but there are only a few reports in which the mechanism(s) of natural drug resistance of SCLC and NSCLC has been analyzed. Etoposide and doxorubicin are thought to be among the most efficacious agents against lung cancer. DNA topoisomerase II, which cleaves the tangling doublestrand DNA and rejoins it, is thought to be a target enzyme of these agents (3). Etoposide and doxorubicin may stabilize transient double-strand breaks consisting of DNA and the enzyme (cleavable complex) (4). This stabilization of the cleavable complex is thought to be the main cause for the cytotoxic effect of topoisomerase Il-reactive agents. Per et al. (5) showed that low topoisomerase II activity and decreased amounts of topoisomerase II induced resistance to amsacrine in amsacrine-resistant P388 cells. Tan et al. (6) reported that the RajiHN2 cell line, a subline resistant to nitrogen mustard, was hypersensitive and had a higher topoisomerase II content and an increased transcription of the topoisomerase II gene compared with those of Raji cells. Glisson et al. (7) reported no significant difference in catalytic activities between a Chinese hamster ovary cell line resistant to etoposide and its wild-type cell line. They suggested that a lack of drug-stimulated DNA cleavage might contribute to the difference in the sensitivities to etoposide. Thus, quantitative and qualitative changes in topoisomerase II may be important factors that determine the sensitivity of at least some cell types to topoisomerase Il-reactive drugs. Another important factor may be drug accumulation in tumor cells. The multidrug-resistant phenotype is often induced by exposure to doxorubicin and etoposide (8). The etoposide-resistant H69 cell line (H69/VP) of Minato et al. (9) had levels of topoisomerase II activity and enzyme content almost equal to those of the parental H69 cell line. These investigators demonstrated that the main cause of etoposide resistance in H69/VP cells was
decreased drug accumulation mediated by P-glycoprotein (9). In this study, we examined the relationship between sensitivities to topoisomerase Il-reactive agents and the biochemical parameters in human lung cancer cell lines.
Materials and Methods Chemicals. RPMI-1640 medium and calcium-free and magnesium-free Dulbecco's phosphate-buffered saline (PBS) were purchased from Nissui Pharmaceutical Co., Tokyo, Japan. Etoposide was obtained from Bristol-Myers Co., Tokyo. Doxorubicin was purchased from Kyowa Hakko Kogyo Co., Ltd., Tokyo. [3H]Etoposide was obtained from Moravek Biochemicals, Brea, Calif. Kinetoplast DNA (kDNA) was provided by M. Kuwano (Oita Medical School). Other drugs and chemicals were purchased from Sigma Chemical Co., St. Louis, Mo., if not otherwise indicated. Human lung cancer cell lines. The human NSCLC ceil lines PC-7, PC-9, and PC-14 were donated by Y. Hayata (Tokyo Medical College). These cell lines were derived from an adenocarcinoma. The SCLC cell lines Lul30, Lul34-BS, Lul35-T, and Lul39 were donated by T. Terasaki, National Cancer Center Research Institute, Tokyo. H69 and N231, human SCLC cell lines, were established at the National Cancer Institute, Bethesda, Md., and were obtained from Y. Shimosato, National Cancer Center Hospital, Tokyo. To establish cisplatinresistant cell lines, the PC-7/1.0, PC9/0.5, PC-14/1.5, H69/0.4, and N231/0.2 cell lines (the resistant sublines before establishment of the cell lines) were cultured with continuous exposure to cisplatin for 1 year (10). The resultant cell lines had the following increased resistance to cisplatin compared with the parent line: PC-7/CDDP (four times the resistance), PC-9/CDDP (28 times the resistance), PC-14/CDDP (11 times the resistance), H69/CDDP (11 times the resistance), and N231/CDDP (four times the resistance). All cell lines were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Immuno-Biochemical Laboratories, Fujioka, Japan) plus peni-
Downloaded from http://jnci.oxfordjournals.org/ at University of Birmingham on August 27, 2014
difference was statistically significant. Etoposide uptake in SCLC cells was higher than in NSCLC cells; the difference was statistically significant, but this difference may not be sufficient to account for the variation in sensitivities of the cell lines. Topoisomerase II activities of nuclear protein from SCLC cell lines were reproducibly twofold higher than those for NSCLC cell lines. The topoisomerase II content in nuclear protein appeared to be higher in SCLC cell lines than in NSCLC cell lines and corresponded to the sensitivities to doxorubicin and etoposide. In the cisplatin-resistant NSCLC cell lines PC-7/CDDP and PC-14/CDDP, the topoisomerase II content was increased compared with that in the parent lines, but the topoisomerase II content in other cisplatin-sensitive parent lines was similar to that in resistant sublines. Conclusions: These findings suggest that the topoisomerase II activity and content may be major factors in determining sensitivity to topoisomerase II inhibitors. [J Natl Cancer Inst 84:113-118,1992]
Journal of the National Cancer Institute
Vol. 84, No. 2, January 15, 1992
pressed in nanograms of etoposide per milligram of cellular protein. Preparation of nuclear protein. Crude nuclear protein from cells of each cell line was prepared as previously reported by Filipski and Kohn (12). Cells were cultured for 5-21 days, collected, and washed twice with ice-cold nucleus buffer which consisted of 2 mW K2HPO4, 5 mM MgCl2, 150 mM NaCl, 1 mM ethylene glycol-bis(P-aminoethylether) N, N, N\ /V'-tetraacetic acid, and 0.1 mM dithiothreitol, adjusted to pH 6.4. The cells were resuspended in 1 mL of nucleus buffer and 9 mL of nucleus buffer containing 0.35% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride was added. The cell suspension was placed on ice for 15 minutes and then washed with nucleus buffer, which was free of Triton X-100. Nuclear protein was extracted for 1 hour at 4 °C with nucleus buffer containing 0.35 M NaCl. DNA and nuclear debris were pelleted by centrifugation at 17 000g for 15 minutes. All procedures were performed on ice. The protein concentration was determined by the BCA protein assay kit (Pierce Chemical Co., Rockford, 111.). Topoisomerase II activity. Topoisomerase II catalytic activity was assayed by decatenation of kDNA to minicircle DNA, using nuclear protein from cells of each cell line (75). Decatenation was carried out by incubating 5 \iL of nuclear protein with 1 ug of kDNA, 50 mM TrisHC1 (pH 7.5), 85 mM KC1, 10 mM MgCl2, 5 mM dithiothreitol, 0.5 mM of EDTA, 30 |ig of bovine serum albumin, and 1 mM adenosine triphosphate in a final volume of 20 |iL of reaction mixture at 30 °C for 30 minutes. Samples were treated with 5 ^L of stop solution consisting of 5% sodium dodecyl sulfate, 50% glycerol, and 0.05% of bromophenol blue. The samples were electrophoresed through a 1% agarose gel in 40 mM Tris acetate-EDTA (pH 7.2) at 80 V for 3 hours. After being stained with ethidium bromide, the gel was photographed under UV light. Topoisomerase II content. The topoisomerase II content of nuclear protein from cells of each cell line was determined by Western blot analysis using topoisomerase II antiserum. Topoisomerase II antiserum was donated by L. F.
Liu (Johns Hopkins Medical School, Baltimore, Md.). The procedure of Western blot analysis was essentially the same as that described previously (14). In brief, 10 (ig of nuclear protein was electrophoresed on a 7.5% sodium dodecyl sulfate-polyacrylamide slab gel. Proteins on the gel were electrically transferred to a nitrocellulose membrane (Pharmacia LKB Biotechnology, Tokyo). The membranes were plated in 50 mM Tris-400 mM NaCl (pH 7.5) buffer containing 0.05% Tween 20 and rinsed. All rinses were for 45 minutes with three changes of buffer. The incubation with topoisomerase II antiserum was performed for 16 hours at 4 °C. After incubation, the membranes were rinsed and incubated with peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad Laboratories, Richmond, Calif.) in the buffer containing 3% bovine serum albumin. After rinsing, blots were developed using 1-chloro-4-naphthol and H2O2 in the buffer containing 11% methanol. The reaction was stopped by rinsing with water. Statistical methods. The data were analyzed for statistical significance by Student's? test.
Downloaded from http://jnci.oxfordjournals.org/ at University of Birmingham on August 27, 2014
cillin (100 U/mL) and streptomycin (100 Hg/mL). Drug sensitivity. The sensitivity of each cell line to the topoisomerase II inhibitors doxorubicin and etoposide was determined by clonogenic assay described previously (11). All assays were conducted with continuous exposure of the cell lines to drugs. NSCLC cells (3 x 104 PC-7 and PC-7/CDDP cells and 1 x 104 PC-9, PC-9/CDDP, PC-14, and PC14/CDDP cells) were incubated for 7 days. SCLC cells (2 x 105) were incubated for 21 days, except for H69 and H69/CDDP cells (1 x 105), which were incubated for 14 days. For PC-7 and PC7/CDDP cells, the drug concentrations used were 0.003, 0.01, 0.03, or 0.1 ng/mL of doxorubicin and 0.1, 0.3, 1.0, and 3.0 ug/mL of etoposide. For PC-9, PC9/CDDP, PC-14, and PC-14/CDDP cells, drug concentrations were 0.1, 0.3, 1.0, and 3.0 |ig/mL of doxorubicin and 0.3, 1.0, 3.0, or 10.0 fig/mL of etoposide. For SCLC cells, drug concentrations were 0.003, 0.01, 0.03, and 0.1 ng/mL of doxorubicin and 0.03, 0.1, 0.3, and 1.0 ug/mL of etoposide. After incubation, the colonies were counted with a CP-2000 automatic colony counter (Shiraimatsu Instrument, Osaka, Japan). The IC5Q value (i.e., drug concentration inhibiting colony formation to 50% of that in control cultures) was determined graphically from the response curve at three or four drugconcentration points. The relative resistance values for cisplatin-resistant cell lines were determined by the formula: IC50 value of resistant cell line/ICso value of parent cell line. Uptake of etoposide. Net cellular uptake of [3H]etoposide was determined by the method previously described by Glisson et al. (7). In brief, 5 x 106 cells were incubated with 5 \ig/mL of [3H] etoposide at 37 °C for 30 minutes or 60 minutes. After incubation, cells were washed with ice-cold PBS twice. Subsequently, 0.5 mL of 1 M NaOH was added to dissolve the cell pellet, and 0.5 mL 1 M HC1 was added to neutralize the NaOH. The samples were transferred to Aquasol-2 (NEN Research Products, Boston, Mass.), and the radioactivity of the samples was measured with a liquid scintillation counter (LS3801; Beckman Instruments, Inc., Irvine, Calif.). Results were ex-
Results Sensitivity to Topoisomerase II Inhibitors Table 1 shows the sensitivity (IC50) of the human lung cancer cell lines to the topoisomerase II inhibitors doxorubicin and etoposide. The SCLC cell lines were more sensitive than the NSCLC lines, and
Table 1. Characteristics of human lung cancer cell lines* IC50 values, Ug/mL Cell lines
Doubling time, h
NSCLC PC-7 PC-9 PC-14
38 23 20
SCLC Lul30 Lul34-BS Lul35-T Lul39
112 78 86 108
Doxorubicin Etoposide
0.12 0.6 0.8
0.26 3.8 2.3
0.014 0.019 0.012 0.009
ND 0.14 0.11 ND
*NSCLCs were adenocarcinomas. ND = not determined.
REPORTS
115
20 40 Exposure Time (min)
Uptake of Etoposide Fig. 1 shows radioactive accumulation in lung cancer cell lines after incubation with 5 ng/mL of [3H]etoposide. We compared the accumulation in SCLC cell lines and NSCLC cell lines in Fig. 1A. Net uptakes of [3H]etoposide were higher in SCLC cell lines than in NSCLC cell lines, and the difference was statistically significant (P
targets. Thus, the use of IL-2 may represent a uniquely attractive approach to enhance MAb penetration into tumor sites and render MAb therapy more effective.
(16) PEACE
DJ.
CHEEVER
MA:
Toxicity
and
therapeutic efficacy of high-dose interleukin 2. In vivo infusion of antibody to NK-1.1 attenuates toxicity without compromising efficacy against murine leukemia. J Exp Med 169:161-173.1989 (17) ORTALDO JR, MASON A, OVERTON R: Lym-
References
phokine-activated killer cells. Analysis of progenitors and effectors. J Exp Med 164:1193-1205.1986
(/) BERINSTEIN N. LEVY R: Treatment of a murine
B cell lymphoma with monoclonal antibodies and IL-2. J Immunol 139:971-976. 1987 (2) BERINSTEIN N. STARNES CO. LEVY R: Specific
enhancement of the therapeutic effect of antiidiotype antibodies on a murine B cell lymphoma by IL-2. J Immunol 140:2839-2845. 1988 (3) SCHULTZ KR. KLARNET JP, PEACE DJ. ET AL:
Monoclonal antibody therapy of murine lymphoma: Enhanced efficacy by concurrent administration of interleukin 2 or lymphokineactivated killer cells. Cancer Res 50:5421 — 5425. 1990
Determinants of Response to the DNA Topoisomerase II Inhibitors Doxorubicin and Etoposide in Human Lung Cancer Cell Lines
(4) PRESS OW, EARY JF, BADGER CC. ET AL:
Treatment of refractory non-Hodgkins lymphoma with radiolabeled MB-1 (anti-CD37) antibody. J Clin Oncol 7:1027-1038. 1989 (5) ROSENSTEIN M, ETTINCHAUSEN SE. ROSENBERG
SA: Extravasation of intravascular fluid mediated by the systemic administration of recombinant interleukin-2. J Immunol 137:1735-1742,1986 (6) DAMLE NK. DOYLE LV. BENDER JR. ET AL: In-
Kazuo Kasahara, Yasuhiro Fujiwara, Yoshikazu Sugimoto, Kazuto Nishio, Tomohide Tamura, Tamotsu Matsuda, Nagahiro Saijo*
terleukin 2-activated human lymphocytes exhibit enhanced adhesion to normal vascular endothelial cells and cause their lysis. J Immunol 138:1779-1785, 1987 (7) SCULIER JP, BODY JJ, NEJAI S, ET AL: En-
dogenous production of tumor necrosis factoralpha in cancer patients during adoptive immunotherapy with interleukin-2 (IL-2). Proc AACR 29:4093, 1988 (8) LOTZE MT. MATORY YL. ETTINGHAUSEN SE.
ET AL: In vivo administration of purified human interleukin 2. II. Half life, immunologic effects, and expansion of peripheral lymphoid cells in vivo with recombinant IL-2. J Immunol 135:2865-2876, 1985 (9) BADGER CC, KROHN KA, PETERSON AV, ET
AL: Experimental radiotherapy of murine lymphoma with l3lI-labeled anti-Thy I.I monoclonal antibody. Cancer Res 45:1536-1544, 1985 (10) NOWINSKI RC, HAYS EF, DOYLE T, ET AL: On-
comaviruses produced by murine leukemia cells in culture. Virology 81:363-370, 1977 (//)
DENKERS EY, BADGER CC, LEDBETTER JA, ET
AL: Influence of antibody isotype on the passive serotherapy of lymphoma. J Immunol 135:2183-2186,1985 (12) BRADFORD MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254, 1976 (13) COLTON T, ED: Statistics in Medicine. Boston: Little, Brown, 1974 (14) ROSENBERG SA, LOTZE MT, MUUL LM, ET AL:
Observations on the systemic administration of autologous lymphokine-activated killer cells and recombinant interleukin-2 to patients with metastatic cancer. N Engl J Med 313:1485— 1492,1985 (15) GRIMM EA, MAZUMDER A, ZHANG HS, ET AL:
Lymphokine-activated killer cell phenomenon. Lysis of natural killer-resistant fresh solid tumor cells by interleukin 2-activated autologous human peripheral blood lymphocytes. J Exp Med 155:1823-1841, 1982
Background: Small-cell lung cancer (SCLC) is more sensitive to anticancer agents than non-small-cell lung cancer (NSCLC), but few studies have analyzed the mechanisms of natural drug resistance responsible for this difference. Purpose: To elucidate these mechanisms, we determined drug sensitivity and evaluated the biochemical parameters affecting response to the DNA topoisomerase II inhibitors doxorubicin and etoposide in both types of cancer cell lines, in particular the activity and content of DNA topoisomerase II, as well as etoposide uptake and cell doubling time. Methods: Drug sensitivity and cellular uptake of etoposide were determined by clonogenic assay and accumulation of radiolabeled drug, respectively. The topoisomerase II activity was assayed by decatenation of kinetoplast DNA to minicircle DNA using nuclear protein, and the content was determined by immunoblot analysis of nuclear extracts. We also compared the topoisomerase II content in parent cell lines with that in lines with cisplatin resistance acquired in vitro. Results: Sensitivities to doxorubicin and etoposide were higher in SCLC cell lines than in NSCLC lines, and the REPORTS 113
Downloaded from http://jnci.oxfordjournals.org/ at University of Birmingham on August 27, 2014
cells in vitro (6) and may cause endothelial damage in vivo resulting in extravasation of intravascular fluid. TNF-a and IFN-y (14,15), released by LAK cells, may also contribute to increased capillary permeability. The effects of IL-2 in tumors are even more complex, since augmentation of the Fc receptor-bearing LAK cell population with the natural killer phenotype has been shown to be the main population of LAK cells causing capillary leak in vivo (16,17). This population can lyse tumor tissues through the mechanism of antibody-dependent cellular cytotoxicity, which may affect subsequent antibody uptake in tumors. IL-2-induced LAK cells may also lyse tumor cells, leading to release of Thy 1 antigen and formation of antibody-antigen complexes. Either of these two mechanisms may result in decreased accumulation of MAb at the tumor site. The efficacy of combined IL-2 and MAb therapy might be enhanced by changing the MAb-dosing schedule in relationship to IL-2 administration, since this maneuver could potentially increase the localization of MAb at the tumor site by taking advantage of a time period of greater capillary permeability induced by high-dose IL-2. Additionally, our data demonstrated similar biodistribution of MAb and albumin in IL-2-treated hosts (Figs. 1, 2). Consequently, approaches using smaller molecules, such as F(ab')2 fragments, could result in more selective extravasation of the MAb without the extracellular edema associated with the present regimen. Our results strongly suggest that IL-2 therapy can enhance the delivery of a radioisotope to a tumor site via a tumorspecific MAb. Moreover, since high doses of IL-2 cause activation of in vivo LAK effector cells, which are able to mediate antibody-dependent cellular cytotoxicity in vitro (1-3), the combination of IL-2 and MAb might also prove beneficial for therapy using nonradiolabeled MAb. Using lower nontoxic doses of IL-2 in combination with 1A14 MAb, we (3) have previously reported augmented antibody-induced regression of tumors. The use of higher doses of IL-2 may not only induce Fc receptor-positive antibody-dependent cellular cytotoxicity LAK effector cells, but may also result in increased amounts of MAb on the tumor
Lung cancer consists of two phenotypically distinct forms: small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC). One distinctive difference between SCLC and NSCLC is chemosensitivity. SCLC is more sensitive
Received May 23, 1991; revised November 7, 1991; accepted November 13, 1991. Supported by Grants-in-Aid from the Ministry of Health and Welfare, Japan, for the Comprehensive 10-Year Strategy for Cancer Control. K. Kasahara, Pharmacology Division. National Cancer Center Research Institute, Tokyo. Japan, and Department of Third Internal Medicine. Kanazawa University, School of Medicine, Kanazawa, Japan. Y. Fujiwara, Y. Sugimoto, K. Nishio, N. Saijo. Pharmacology Division, National Cancer Center Research Institute. T. Tamura, Department of Internal Medicine. National Cancer Center Hospital, Tokyo. T. Matsuda, Department of Third Internal Medicine, Kanazawa University, School of Medicine. We thank John Stephan Lazo. Department of Pharmacology. Pittsburgh University, for his critical review of the manuscript. *Correspondence to: Nagahiro Saijo. M.D.. Pharmacology Division, National Cancer Center Research Institute. Tsukiji 5-1-1, Chuo-ku. Tokyo 104, Japan.
114
to anticancer agents than NSCLC (/). Nakagawa et al. (2) reported that expression of glutathione S-transferase n messenger RNA in three SCLC cell lines and three NSCLC cell lines inversely correlated with sensitivity to cisplatin and carboplatin, but there are only a few reports in which the mechanism(s) of natural drug resistance of SCLC and NSCLC has been analyzed. Etoposide and doxorubicin are thought to be among the most efficacious agents against lung cancer. DNA topoisomerase II, which cleaves the tangling doublestrand DNA and rejoins it, is thought to be a target enzyme of these agents (3). Etoposide and doxorubicin may stabilize transient double-strand breaks consisting of DNA and the enzyme (cleavable complex) (4). This stabilization of the cleavable complex is thought to be the main cause for the cytotoxic effect of topoisomerase Il-reactive agents. Per et al. (5) showed that low topoisomerase II activity and decreased amounts of topoisomerase II induced resistance to amsacrine in amsacrine-resistant P388 cells. Tan et al. (6) reported that the RajiHN2 cell line, a subline resistant to nitrogen mustard, was hypersensitive and had a higher topoisomerase II content and an increased transcription of the topoisomerase II gene compared with those of Raji cells. Glisson et al. (7) reported no significant difference in catalytic activities between a Chinese hamster ovary cell line resistant to etoposide and its wild-type cell line. They suggested that a lack of drug-stimulated DNA cleavage might contribute to the difference in the sensitivities to etoposide. Thus, quantitative and qualitative changes in topoisomerase II may be important factors that determine the sensitivity of at least some cell types to topoisomerase Il-reactive drugs. Another important factor may be drug accumulation in tumor cells. The multidrug-resistant phenotype is often induced by exposure to doxorubicin and etoposide (8). The etoposide-resistant H69 cell line (H69/VP) of Minato et al. (9) had levels of topoisomerase II activity and enzyme content almost equal to those of the parental H69 cell line. These investigators demonstrated that the main cause of etoposide resistance in H69/VP cells was
decreased drug accumulation mediated by P-glycoprotein (9). In this study, we examined the relationship between sensitivities to topoisomerase Il-reactive agents and the biochemical parameters in human lung cancer cell lines.
Materials and Methods Chemicals. RPMI-1640 medium and calcium-free and magnesium-free Dulbecco's phosphate-buffered saline (PBS) were purchased from Nissui Pharmaceutical Co., Tokyo, Japan. Etoposide was obtained from Bristol-Myers Co., Tokyo. Doxorubicin was purchased from Kyowa Hakko Kogyo Co., Ltd., Tokyo. [3H]Etoposide was obtained from Moravek Biochemicals, Brea, Calif. Kinetoplast DNA (kDNA) was provided by M. Kuwano (Oita Medical School). Other drugs and chemicals were purchased from Sigma Chemical Co., St. Louis, Mo., if not otherwise indicated. Human lung cancer cell lines. The human NSCLC ceil lines PC-7, PC-9, and PC-14 were donated by Y. Hayata (Tokyo Medical College). These cell lines were derived from an adenocarcinoma. The SCLC cell lines Lul30, Lul34-BS, Lul35-T, and Lul39 were donated by T. Terasaki, National Cancer Center Research Institute, Tokyo. H69 and N231, human SCLC cell lines, were established at the National Cancer Institute, Bethesda, Md., and were obtained from Y. Shimosato, National Cancer Center Hospital, Tokyo. To establish cisplatinresistant cell lines, the PC-7/1.0, PC9/0.5, PC-14/1.5, H69/0.4, and N231/0.2 cell lines (the resistant sublines before establishment of the cell lines) were cultured with continuous exposure to cisplatin for 1 year (10). The resultant cell lines had the following increased resistance to cisplatin compared with the parent line: PC-7/CDDP (four times the resistance), PC-9/CDDP (28 times the resistance), PC-14/CDDP (11 times the resistance), H69/CDDP (11 times the resistance), and N231/CDDP (four times the resistance). All cell lines were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Immuno-Biochemical Laboratories, Fujioka, Japan) plus peni-
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difference was statistically significant. Etoposide uptake in SCLC cells was higher than in NSCLC cells; the difference was statistically significant, but this difference may not be sufficient to account for the variation in sensitivities of the cell lines. Topoisomerase II activities of nuclear protein from SCLC cell lines were reproducibly twofold higher than those for NSCLC cell lines. The topoisomerase II content in nuclear protein appeared to be higher in SCLC cell lines than in NSCLC cell lines and corresponded to the sensitivities to doxorubicin and etoposide. In the cisplatin-resistant NSCLC cell lines PC-7/CDDP and PC-14/CDDP, the topoisomerase II content was increased compared with that in the parent lines, but the topoisomerase II content in other cisplatin-sensitive parent lines was similar to that in resistant sublines. Conclusions: These findings suggest that the topoisomerase II activity and content may be major factors in determining sensitivity to topoisomerase II inhibitors. [J Natl Cancer Inst 84:113-118,1992]
Journal of the National Cancer Institute
Vol. 84, No. 2, January 15, 1992
pressed in nanograms of etoposide per milligram of cellular protein. Preparation of nuclear protein. Crude nuclear protein from cells of each cell line was prepared as previously reported by Filipski and Kohn (12). Cells were cultured for 5-21 days, collected, and washed twice with ice-cold nucleus buffer which consisted of 2 mW K2HPO4, 5 mM MgCl2, 150 mM NaCl, 1 mM ethylene glycol-bis(P-aminoethylether) N, N, N\ /V'-tetraacetic acid, and 0.1 mM dithiothreitol, adjusted to pH 6.4. The cells were resuspended in 1 mL of nucleus buffer and 9 mL of nucleus buffer containing 0.35% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride was added. The cell suspension was placed on ice for 15 minutes and then washed with nucleus buffer, which was free of Triton X-100. Nuclear protein was extracted for 1 hour at 4 °C with nucleus buffer containing 0.35 M NaCl. DNA and nuclear debris were pelleted by centrifugation at 17 000g for 15 minutes. All procedures were performed on ice. The protein concentration was determined by the BCA protein assay kit (Pierce Chemical Co., Rockford, 111.). Topoisomerase II activity. Topoisomerase II catalytic activity was assayed by decatenation of kDNA to minicircle DNA, using nuclear protein from cells of each cell line (75). Decatenation was carried out by incubating 5 \iL of nuclear protein with 1 ug of kDNA, 50 mM TrisHC1 (pH 7.5), 85 mM KC1, 10 mM MgCl2, 5 mM dithiothreitol, 0.5 mM of EDTA, 30 |ig of bovine serum albumin, and 1 mM adenosine triphosphate in a final volume of 20 |iL of reaction mixture at 30 °C for 30 minutes. Samples were treated with 5 ^L of stop solution consisting of 5% sodium dodecyl sulfate, 50% glycerol, and 0.05% of bromophenol blue. The samples were electrophoresed through a 1% agarose gel in 40 mM Tris acetate-EDTA (pH 7.2) at 80 V for 3 hours. After being stained with ethidium bromide, the gel was photographed under UV light. Topoisomerase II content. The topoisomerase II content of nuclear protein from cells of each cell line was determined by Western blot analysis using topoisomerase II antiserum. Topoisomerase II antiserum was donated by L. F.
Liu (Johns Hopkins Medical School, Baltimore, Md.). The procedure of Western blot analysis was essentially the same as that described previously (14). In brief, 10 (ig of nuclear protein was electrophoresed on a 7.5% sodium dodecyl sulfate-polyacrylamide slab gel. Proteins on the gel were electrically transferred to a nitrocellulose membrane (Pharmacia LKB Biotechnology, Tokyo). The membranes were plated in 50 mM Tris-400 mM NaCl (pH 7.5) buffer containing 0.05% Tween 20 and rinsed. All rinses were for 45 minutes with three changes of buffer. The incubation with topoisomerase II antiserum was performed for 16 hours at 4 °C. After incubation, the membranes were rinsed and incubated with peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad Laboratories, Richmond, Calif.) in the buffer containing 3% bovine serum albumin. After rinsing, blots were developed using 1-chloro-4-naphthol and H2O2 in the buffer containing 11% methanol. The reaction was stopped by rinsing with water. Statistical methods. The data were analyzed for statistical significance by Student's? test.
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cillin (100 U/mL) and streptomycin (100 Hg/mL). Drug sensitivity. The sensitivity of each cell line to the topoisomerase II inhibitors doxorubicin and etoposide was determined by clonogenic assay described previously (11). All assays were conducted with continuous exposure of the cell lines to drugs. NSCLC cells (3 x 104 PC-7 and PC-7/CDDP cells and 1 x 104 PC-9, PC-9/CDDP, PC-14, and PC14/CDDP cells) were incubated for 7 days. SCLC cells (2 x 105) were incubated for 21 days, except for H69 and H69/CDDP cells (1 x 105), which were incubated for 14 days. For PC-7 and PC7/CDDP cells, the drug concentrations used were 0.003, 0.01, 0.03, or 0.1 ng/mL of doxorubicin and 0.1, 0.3, 1.0, and 3.0 ug/mL of etoposide. For PC-9, PC9/CDDP, PC-14, and PC-14/CDDP cells, drug concentrations were 0.1, 0.3, 1.0, and 3.0 |ig/mL of doxorubicin and 0.3, 1.0, 3.0, or 10.0 fig/mL of etoposide. For SCLC cells, drug concentrations were 0.003, 0.01, 0.03, and 0.1 ng/mL of doxorubicin and 0.03, 0.1, 0.3, and 1.0 ug/mL of etoposide. After incubation, the colonies were counted with a CP-2000 automatic colony counter (Shiraimatsu Instrument, Osaka, Japan). The IC5Q value (i.e., drug concentration inhibiting colony formation to 50% of that in control cultures) was determined graphically from the response curve at three or four drugconcentration points. The relative resistance values for cisplatin-resistant cell lines were determined by the formula: IC50 value of resistant cell line/ICso value of parent cell line. Uptake of etoposide. Net cellular uptake of [3H]etoposide was determined by the method previously described by Glisson et al. (7). In brief, 5 x 106 cells were incubated with 5 \ig/mL of [3H] etoposide at 37 °C for 30 minutes or 60 minutes. After incubation, cells were washed with ice-cold PBS twice. Subsequently, 0.5 mL of 1 M NaOH was added to dissolve the cell pellet, and 0.5 mL 1 M HC1 was added to neutralize the NaOH. The samples were transferred to Aquasol-2 (NEN Research Products, Boston, Mass.), and the radioactivity of the samples was measured with a liquid scintillation counter (LS3801; Beckman Instruments, Inc., Irvine, Calif.). Results were ex-
Results Sensitivity to Topoisomerase II Inhibitors Table 1 shows the sensitivity (IC50) of the human lung cancer cell lines to the topoisomerase II inhibitors doxorubicin and etoposide. The SCLC cell lines were more sensitive than the NSCLC lines, and
Table 1. Characteristics of human lung cancer cell lines* IC50 values, Ug/mL Cell lines
Doubling time, h
NSCLC PC-7 PC-9 PC-14
38 23 20
SCLC Lul30 Lul34-BS Lul35-T Lul39
112 78 86 108
Doxorubicin Etoposide
0.12 0.6 0.8
0.26 3.8 2.3
0.014 0.019 0.012 0.009
ND 0.14 0.11 ND
*NSCLCs were adenocarcinomas. ND = not determined.
REPORTS
115
20 40 Exposure Time (min)
Uptake of Etoposide Fig. 1 shows radioactive accumulation in lung cancer cell lines after incubation with 5 ng/mL of [3H]etoposide. We compared the accumulation in SCLC cell lines and NSCLC cell lines in Fig. 1A. Net uptakes of [3H]etoposide were higher in SCLC cell lines than in NSCLC cell lines, and the difference was statistically significant (P
