Archives of
Arch GynecolObstet (1992) 251:79-86
Gynecology
and Obstetrics © Springer-Verlag 1992
Expression of a human multidrug resistance gene in human ovarian carcinoma cell lines S. Sekiya 1, T. Nunoyama l, H. Shirasawa 2, H. Kimura 1, M. Kawata 1, N. Iijima 1, Y. Sugimoto 3, T. Tsuruo 4, and H. Takamizawa 1 1Department of Obstetrics and Gynecologyand 2Department of 1st Microbiology,Chiba University School of Medicine, 1-8-1 Inohana, Chiba 280, 3Divisionof Experimental Chemotherapy, Cancer ChemotherapyCenter, Japanese Foundationfor Cancer Research, KamiIkebukuro, Toshima-ku,Tokyo 170, and 4Instituteof Applied Microbiology,Universityof Tokyo, Yayoi, Bunkyoku,Tokyo 113, Japan Received March 6, 1991/AcceptedNovember 5, 1991
Summary. To investigate the possible role of the multidrug resistance phenotype to chemoresistance in human ovarian carcinoma, we have analyzed human multidrug resistance gene (mdr 1) expression in 8 human ovarian adenocarcinoma cell lines. An increase in P-glycoprotein level specific to multidrugresistant tumor cells was not apparently associated with the increase in resistance to vincristine (VCR) or doxorubicin (Adriamycin). Mdr 1 transcripts (4.5 kilobases) were observed in the R N A preparation obtained from only one cell line (SHIN-3) that showed the highest resistance to both drugs in vitro and in vivo. No cell lines showed mdr 1 DNA amplification. These results suggest that the insensitivity of human ovarian carcinoma to chemotherapy could be partly explained by the expression of mdr 1. Key words: Multidrug resistance - Ovarian carcinoma - Mdr 1 gene P-glycoprotein
Introduction Involvement of the multidrug resistant phenotype in acquired chemoresistance has been studied extensively in tumor cells grown in tissue culture and selected for resistance to various chemotherapeutic agents. The multidrug resistant cells display cross-resistance to a broad range of drugs, such as Vinca alkaloids, anthracyclines, etoposide and actinomycin D. The multidrug resistant phenotype is correlated with over-expression of the human multidrug resistant gene (mdr 1) which codes for the transmembrane protein P-glycoprotein, and with increased drug effiux [1-3]. Tumor cells with significant levels of P-glycoprotein have been identified in a variety of human tumors. However, the question of whether the presence of multidrug-resistant tumor cells containing P-glycoprotein limits the clinical response of ovarian carcinoma to chemotherapy remains Offprint requests to:
Dr. S. Sekiya(address see above)
80
S. Sekiya et al.
to be answered. To clarify the role of the multidrug resistance phenotype with respect tao chemotherapy in ovarian carcinoma, we have examined correlation between the level of resistance to VCR or Adriamycin in vitro and in vivo and multidrug resistance phenotype expression using 8 human ovarian carcinoma cell lines.
Materials and Methods Cell lines Eight kinds of human ovarian carcinoma cell lines independently derived from epithelial adenocarcinomas were used (Table 1). Cell biological characteristics of the cell lines were reported elsewhere [4]. Cell monolayers were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (Flow Lab., Sydney, Australia) and antibiotics (penicillin, 100 units/ml, and streptomycin, 100 gg/ml) under a humidified atmosphere of 5% CO2 and 95% air at 37°C.
Drugs Vincristine sulfate and doxorubicin hydrochloride were purchased from the Sigma Chemical Co. (St. Louis, MO). Stocks of VCR or Adriamycin (100 ~tg/ml) were prepared by solution in serum-free medium and frozen at - 2 0 ° C in the dark until use. Table 1. Human ovarian adenocarcinoma cell lines used and their growth capacity in vitro and in vivo Origin
Cell line
Growth capacity In vitro
In vivo
Colony forming efficiencya
Population doubling time b
(%)
(h)
Tumorigenicityc
Volume doubling time d
(h)
Serous cystadenocarcinoma
HOC-21 SHIN-3 HUOA
15 48 5
22 22 52
5/5e 5/5 0/5
135 76 --
Mucinous cystadenocarcinoma
OMC-3
5
82
5/5
83
Endometrioid adenocarcinoma
HOC-I HMOA
2 23
38 46
2/5 0/5
---
Mesonephroid adenocarcinoma
HAC-2 HUOCA-II
19 6
54 60
5/5 5/5
40 24
a Cells (500 to 1,000) were plated in 25-cmz flasks and macroscopic colonies (32 cells) were counted on the 14th day of incubation. b Assessed for ceils in exponential phase of growth after plating 10s cells in 25-cm 2 flasks. c Cells (5 x 106) were inoculated subcutaneously into the femoral region of 6-week-old female Balb/ c nude mice. d Assessed for tumors in exponential phase of growth. No. of tumor/No, of nude mice inoculated
Multidrug resistance in ovarian carcinoma
81
Measurement of drug sensitivity in vitro and in vivo The sensitivity of human ovarian carcinoma cell lines to VCR or Adriamycin in vitro was measured using a colony formation assay. Cells (500 to 1,000) were plated in duplicate in 2 ml of culture medium in Falcon 35-ram multiwell plate (Becton Dickinson & Co., Lincoln, NJ) and incubated for 24 h. Then cells were exposed for 1 h to VCR or Adriamycin at various concentrations, washed with serum-free medium and further incubated for 14 days. Macroscopic colonies consisting of more than 32 cells were counted after fixation with methanol and Giemsa staining. For in vivo measurement of drug sensitivity, cells (5 x 106) were inculated subcutaneously into the inguinal region of 6-week-old female Balb/c nude mice (Clea Japan Inc., Tokyo, Japan). When the tumor volume (length x width2/2) reached 102 to 103 mm 3, VCR (1 mg/kg) or Adriamycin (3 mg/kg) was injected into the intraperitoneal cavity once a week for 5 to 7 weeks.
Indirect immunofluorescence staining with monoclonal antibody to P-glycoprotein Cells were fixed in 1% paraformaldehyde/phosphate-buffered saline solution (PBS) for 30 min at 4°C. They were then washed with PBS, and incubated for 30 min at 4°C with 0.2 ml of monoclonal antibody to P-glycoprotein (MRK 16) [5] containing 2 ~tg of protein. They were washed and incubated for an additional 30 min at 4°C with 0.2 ml of fluorescein-conjugated IgG fraction of goat anti-mouse immunoglobulins (Cappel, Cochranville, PA). After a second wash with PBS, the cells were analyzed using flow cytometry with a FACStar (Becton Dickinson, Mountain View, CA).
Isolation of DNA and RNA Cellular DNA was extracted using phenol/chloroform, and total cellular RNA was prepared by the guanidium thiocynate/caesium chloride method as had been described [6].
DNA and RNA blot analyses For Southern blotting, DNAs extracted from cells were digested with an excess amount of EcoRI and electrophoresed on a 0.8% agarose gel. The separated DNAs were denaturated, neutralized and transferred to a nitrocellulose filter overnight. For Northern blotting, total RNAs from cells were denaturated at 60°C for 15 min in 6.3% formaldehyde, 50% formamide, 5 mM MOPS (pH 7.0), 15 mM sodium acetate, 1 mM EDTA. The denaturated RNAs were separated on a 1.0% agarose gel containing 6.3% formaldehyde, 20 mM MOPS (pH 7.0), 15 mM sodium acetate, i mM EDTA. The separated RNAs were transfered to a nitrocellulose filter overnight. The blotted filters were baked at 80°C for 2 h. Hybridization with 32P-labelled cDNA of P-glycoprotein [7, 8] and autoradiography were carried out as had been described [6]. Amplification and transcription levels of the mdr 1 gene were measured by a densitometer.
Results T h e in v i t r o a n d in v i v o g r o w t h c a p a c i t y o f 8 h u m a n o v a r i a n a d e n o c a r c i n o m a cell l i n e s is s u m m a r i z e d in T a b l e 1. T h e c a p a c i t y d i f f e r e d b e t w e e n cell lines. S e n s i t i v i t y o f cell lines to V C R o r A d r i a m y c i n in v i t r o is e x p r e s s e d as a 5 0 % r e d u c t i o n in cell c o l o n y n u m b e r (ICs0) (Figs. 1 a n d 2) a n d s u m m a r i z e d in T a b l e 2. T h e r e w a s g o o d c o r r e l a t i o n b e t w e e n t h e s e n s i t i v i t y o f cells to V C R a n d A d r i a m y c i n . T h i s i n d i c a t e s t h a t t h e m u l t i d r u g r e s i s t a n c e is e x p r e s s e d in t h e cell l i n e s t h a t s h o w e d l o w s e n s i t i v i t y to t h e d r u g s . T h e l e v e l s o f m u l t i d r u g r e s i s t a n c e a m o n g t h e 8 cell lines in v i t r o w e r e as f o l l o w s ; S H I N - 3 ( h i g h l y r e s i s t a n t g r o u p )
S. Sekiya et at.
82
100
SHIN-3
HAG-2
50-
L_
I00-
Hoe-21
50-
m
~"
t__
lOO
HOC~I
50-
100
HMOA
HUOCA-]
'1 i i
030.5 I
5 &l 0.5 1 Concentration
lO0
50-
100-
50-
I00-
50-
Fig. L Sensitivity of 8 h u m a n ovarian adenocarcinoma cell lines to vincristine in vitro. See Materials and Methods
~ ~ ~
-3
(/tg/m~,)
HUOA
\ Hoe-!
-2
A
100"
50-~-3
HOC-21
Fig. 2. Sensitivity of 8 h u m a n ovarian adenocarcinoma cell lines to adriamycin in vitro
o.i o'.5 i
5 o.~o.s C o n c e n t r a t i o n (pg/m~.)
Multidrug resistance in ovarian carcinoma
83
Table 2. Levels of P-glycoprotein and its mRNA and DNA amplification in 8 human ovarian adenocarcinoma cell lines Cell line
IC50~ Vincristine (~tg/ml)
Adriamycin (p.g/mt)
CMK ~ K 562~
NT~ NT
NT NT
SHIN-3 OMC-3 HUOA HMOA HAC-2 HOC-21 HOC-I HUOCA-II
>5 1.3 1.1 0.2 0.2 0.1 --, non-treated; - - e - - - , treated) or adriamycin (--A--, non-treated; - - A - - treated) on the growth of tumors developed by inoculation of 3 human ovarian adenocarcinoma cell lines in vivo. I indicates the death of nude mouse. See Materials and Methods
Weeks after inoculation
1
4.5kb
2
34
5
6
7 8 9
--
Fig. 4. Mdr 1 RNA expression in 8 human ovariand adenocarcinoma cell lines by a Northern blot. Lane 1, HeLa (negative control for mdr 1 RNA transcripts); Lane 2, HOC-21; Lane 3, SHIN-3 (positive) ; Lane 4, HUOA; Lane 5, OMC-3; Lane 6, HMOA; Lane 7, HOC-I; Lane 8, HAC-2; Lane 9, HUOCA-II. kb, kilobase
Multidrug resistance in ovarian carcinoma
85
Discussion
Detection of P-glycoprotein in ovarian carcinoma was first reported by Bell et al. [10] in 1985. A monoclonal antibody against P-glycoprotein was used to examine tumor samples from 5 patients with advanced ovarian carcinoma. High levels of P-glycoprotein (a molecular weight 170 kilodalton membrane component) were detected by using an immunoblotting method in samples from 2 patients who had undergone intensive chemotherapy. Bourhis et al. [11] analyzed mdr 1 gene expression in fresh ovarian carcinoma specimens from 50 patients by Northern and slot blot hybridization techniques using a mdr 1 cDNA probe. Mdr 1 transcripts, possibly the same ones observed in the present study, were detected in RNA preparations obtained from 8 of 10 patients who had been treated with VCR and/or Adriamycin. In contrast to these reports, Volm et al. [12] recently reported that the P-glycoprotein could be detected in ovarian carcinomas not previously treated with chemotherapy (one positive of 5 patients not previously treated). In parallel with these studies on human ovarian carcinoma, isolation of drugresistant derivatives from drug-sensitive parent human ovarian carcinoma cell lines by continuous exposure to increasing concentrations of VCR in vitro has been reported [13, 14]. The acquired multidrug resistant derivates showed extremely high ranges in resistance (more than 1,000-fold) and demonstrated corresponding overexpression of P-glycoprotein at both protein and mRNA levels. As compared with these in vitro models, range in resistance appeared to be low in our human ovarian carcinoma cell lines and the levels to be similar to those in ovarian carcinoma specimens. In our present results, only one (SHIN3) of 8 cell lines that appeared to be multidrug resistant in vitro and in vivo showed mdr 1 transcripts with a slight increase of P-glycoprotein. No cell lines showed the amplification of mdr 1 DNA. Bradley et al. [15] also reported an overexpression of mdr 1 gene with no DNA amplification in a multidrugresistant human ovarian carcinoma cell line. This weak multidrug resistance process appears to be characteristics of ovarian carcinoma and gene expression of the multidrug resistance phenotype is probably under complex control in ovarian carcinoma. Acknowledgement~ We thank Dr. T. Sato, Department of Pediatrics, Chiba University School of Medicine, for the gift of the CMK cell line.
References I. Gerlach J, Kartner N, Bell DR, Ling V (1986) Multidrug resistance. Cancer Surv 5:25-46 2. Gottesman MM, Pastan I (1988) Resistance to multiple chemotherapeutic agents in human cancer cells. TIPS 9:54-58 3. Tsuruo T (1988) Mechanisms of multidrug resistance and implications for therapy. Jpn J Cancer Res (Gann) 79:285-296 4. Sekiya S, Tanaka N, Takamizawa H (1990) Production of various marker substances in human ovarian cancer cell lines of epithelial origin. Arch Gynecol Obstet 248:103-110
86
S. Sekiya et al.
5. Hamada H, Tsuruo T (1986) Functional role of the 170- to 180-kDa glycoprotein specific to drugresistant tumor cells as revealed by monoclonal antibodies. Proc Natl Acad Sci USA 83:7758-7789 6. Shirasawa H, Tomita Y, Sekiya S, Takamizawa H, Shimizu B (1987) Integratiion and transcription of human papillomavirus type 16 and 18 sequences in cell lines derived from cervical carcinomas. J Gen Virol 68:583-591 7. Sugimoto Y, Tsuruo T (1987) DNA-mediated transfer and cloning of a human multidrugresistant gene of adriamycin-resistant myelogenous leukemia K 562. Cancer Res 47:2620-2625 8. Sugimoto Y, Asami N, Tsuruo T (1989) Expression of P-glycoprotein mRNA in human gastric tumors. Jpn J Cancer Res (Gann) 80:993-999 9. Ishida Y, Ohtsu T, Hamada H, Sugimoto Y, Tobinai K, Minato K, Tsuruo T, Shimoyama M (t989) Multidrug resistance in cultured human leukemia and lymphoma cell lines detected by a monoclonal antibody, MRK 16. Jpn J Cancer Res (Gann) 80:1006-1013 10. Bell DR, Gerlach JH, Kartner N, Buick RN, Ling V (1985) Detection of P-glycoprotein in ovarian cancer: a molecular marker associated with multidrug resistance. J Clin Oncol 3:311315 11. Bourhis J, Goldstein LJ, Riou G, Pastan I, Gottesman MM, Benard J (1989) Expression of a human multidrug gene in ovarian carcinomas. Cancer Res 49:5062-5065 12. Volm M, Efferth T, Bak M, Mattern J (1989) Detection of drug resistance in human ovarian carcinoma. Arch Gynecol Obstet 244:123-128 13. Chan HSL, Bradley G, Thorner P, Haddad G, Gallie BL, Ling V (1988) A sensitive method for immunocytochemical detection of P-glycoprotein in multidrug-resistant human ovarian carcinoma cell lines. Lab Invest 59:870-875 14. Bernard J, Da Silva J, Teyssier J-R, Riou G (1989) Over-expression of mdr 1 gene with no DNA amprification in a multiple-drug-resistant human ovarian carcinoma cell line. Int J Cancer 43:471-477 15. Bradley G, Naik M, Ling V (1989) P-glycoprotein expression in multi-drug-resistant human ovarian carcinoma cell lines. Cancer Res 49:2790-2796
Arch GynecolObstet (1992) 251:79-86
Gynecology
and Obstetrics © Springer-Verlag 1992
Expression of a human multidrug resistance gene in human ovarian carcinoma cell lines S. Sekiya 1, T. Nunoyama l, H. Shirasawa 2, H. Kimura 1, M. Kawata 1, N. Iijima 1, Y. Sugimoto 3, T. Tsuruo 4, and H. Takamizawa 1 1Department of Obstetrics and Gynecologyand 2Department of 1st Microbiology,Chiba University School of Medicine, 1-8-1 Inohana, Chiba 280, 3Divisionof Experimental Chemotherapy, Cancer ChemotherapyCenter, Japanese Foundationfor Cancer Research, KamiIkebukuro, Toshima-ku,Tokyo 170, and 4Instituteof Applied Microbiology,Universityof Tokyo, Yayoi, Bunkyoku,Tokyo 113, Japan Received March 6, 1991/AcceptedNovember 5, 1991
Summary. To investigate the possible role of the multidrug resistance phenotype to chemoresistance in human ovarian carcinoma, we have analyzed human multidrug resistance gene (mdr 1) expression in 8 human ovarian adenocarcinoma cell lines. An increase in P-glycoprotein level specific to multidrugresistant tumor cells was not apparently associated with the increase in resistance to vincristine (VCR) or doxorubicin (Adriamycin). Mdr 1 transcripts (4.5 kilobases) were observed in the R N A preparation obtained from only one cell line (SHIN-3) that showed the highest resistance to both drugs in vitro and in vivo. No cell lines showed mdr 1 DNA amplification. These results suggest that the insensitivity of human ovarian carcinoma to chemotherapy could be partly explained by the expression of mdr 1. Key words: Multidrug resistance - Ovarian carcinoma - Mdr 1 gene P-glycoprotein
Introduction Involvement of the multidrug resistant phenotype in acquired chemoresistance has been studied extensively in tumor cells grown in tissue culture and selected for resistance to various chemotherapeutic agents. The multidrug resistant cells display cross-resistance to a broad range of drugs, such as Vinca alkaloids, anthracyclines, etoposide and actinomycin D. The multidrug resistant phenotype is correlated with over-expression of the human multidrug resistant gene (mdr 1) which codes for the transmembrane protein P-glycoprotein, and with increased drug effiux [1-3]. Tumor cells with significant levels of P-glycoprotein have been identified in a variety of human tumors. However, the question of whether the presence of multidrug-resistant tumor cells containing P-glycoprotein limits the clinical response of ovarian carcinoma to chemotherapy remains Offprint requests to:
Dr. S. Sekiya(address see above)
80
S. Sekiya et al.
to be answered. To clarify the role of the multidrug resistance phenotype with respect tao chemotherapy in ovarian carcinoma, we have examined correlation between the level of resistance to VCR or Adriamycin in vitro and in vivo and multidrug resistance phenotype expression using 8 human ovarian carcinoma cell lines.
Materials and Methods Cell lines Eight kinds of human ovarian carcinoma cell lines independently derived from epithelial adenocarcinomas were used (Table 1). Cell biological characteristics of the cell lines were reported elsewhere [4]. Cell monolayers were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (Flow Lab., Sydney, Australia) and antibiotics (penicillin, 100 units/ml, and streptomycin, 100 gg/ml) under a humidified atmosphere of 5% CO2 and 95% air at 37°C.
Drugs Vincristine sulfate and doxorubicin hydrochloride were purchased from the Sigma Chemical Co. (St. Louis, MO). Stocks of VCR or Adriamycin (100 ~tg/ml) were prepared by solution in serum-free medium and frozen at - 2 0 ° C in the dark until use. Table 1. Human ovarian adenocarcinoma cell lines used and their growth capacity in vitro and in vivo Origin
Cell line
Growth capacity In vitro
In vivo
Colony forming efficiencya
Population doubling time b
(%)
(h)
Tumorigenicityc
Volume doubling time d
(h)
Serous cystadenocarcinoma
HOC-21 SHIN-3 HUOA
15 48 5
22 22 52
5/5e 5/5 0/5
135 76 --
Mucinous cystadenocarcinoma
OMC-3
5
82
5/5
83
Endometrioid adenocarcinoma
HOC-I HMOA
2 23
38 46
2/5 0/5
---
Mesonephroid adenocarcinoma
HAC-2 HUOCA-II
19 6
54 60
5/5 5/5
40 24
a Cells (500 to 1,000) were plated in 25-cmz flasks and macroscopic colonies (32 cells) were counted on the 14th day of incubation. b Assessed for ceils in exponential phase of growth after plating 10s cells in 25-cm 2 flasks. c Cells (5 x 106) were inoculated subcutaneously into the femoral region of 6-week-old female Balb/ c nude mice. d Assessed for tumors in exponential phase of growth. No. of tumor/No, of nude mice inoculated
Multidrug resistance in ovarian carcinoma
81
Measurement of drug sensitivity in vitro and in vivo The sensitivity of human ovarian carcinoma cell lines to VCR or Adriamycin in vitro was measured using a colony formation assay. Cells (500 to 1,000) were plated in duplicate in 2 ml of culture medium in Falcon 35-ram multiwell plate (Becton Dickinson & Co., Lincoln, NJ) and incubated for 24 h. Then cells were exposed for 1 h to VCR or Adriamycin at various concentrations, washed with serum-free medium and further incubated for 14 days. Macroscopic colonies consisting of more than 32 cells were counted after fixation with methanol and Giemsa staining. For in vivo measurement of drug sensitivity, cells (5 x 106) were inculated subcutaneously into the inguinal region of 6-week-old female Balb/c nude mice (Clea Japan Inc., Tokyo, Japan). When the tumor volume (length x width2/2) reached 102 to 103 mm 3, VCR (1 mg/kg) or Adriamycin (3 mg/kg) was injected into the intraperitoneal cavity once a week for 5 to 7 weeks.
Indirect immunofluorescence staining with monoclonal antibody to P-glycoprotein Cells were fixed in 1% paraformaldehyde/phosphate-buffered saline solution (PBS) for 30 min at 4°C. They were then washed with PBS, and incubated for 30 min at 4°C with 0.2 ml of monoclonal antibody to P-glycoprotein (MRK 16) [5] containing 2 ~tg of protein. They were washed and incubated for an additional 30 min at 4°C with 0.2 ml of fluorescein-conjugated IgG fraction of goat anti-mouse immunoglobulins (Cappel, Cochranville, PA). After a second wash with PBS, the cells were analyzed using flow cytometry with a FACStar (Becton Dickinson, Mountain View, CA).
Isolation of DNA and RNA Cellular DNA was extracted using phenol/chloroform, and total cellular RNA was prepared by the guanidium thiocynate/caesium chloride method as had been described [6].
DNA and RNA blot analyses For Southern blotting, DNAs extracted from cells were digested with an excess amount of EcoRI and electrophoresed on a 0.8% agarose gel. The separated DNAs were denaturated, neutralized and transferred to a nitrocellulose filter overnight. For Northern blotting, total RNAs from cells were denaturated at 60°C for 15 min in 6.3% formaldehyde, 50% formamide, 5 mM MOPS (pH 7.0), 15 mM sodium acetate, 1 mM EDTA. The denaturated RNAs were separated on a 1.0% agarose gel containing 6.3% formaldehyde, 20 mM MOPS (pH 7.0), 15 mM sodium acetate, i mM EDTA. The separated RNAs were transfered to a nitrocellulose filter overnight. The blotted filters were baked at 80°C for 2 h. Hybridization with 32P-labelled cDNA of P-glycoprotein [7, 8] and autoradiography were carried out as had been described [6]. Amplification and transcription levels of the mdr 1 gene were measured by a densitometer.
Results T h e in v i t r o a n d in v i v o g r o w t h c a p a c i t y o f 8 h u m a n o v a r i a n a d e n o c a r c i n o m a cell l i n e s is s u m m a r i z e d in T a b l e 1. T h e c a p a c i t y d i f f e r e d b e t w e e n cell lines. S e n s i t i v i t y o f cell lines to V C R o r A d r i a m y c i n in v i t r o is e x p r e s s e d as a 5 0 % r e d u c t i o n in cell c o l o n y n u m b e r (ICs0) (Figs. 1 a n d 2) a n d s u m m a r i z e d in T a b l e 2. T h e r e w a s g o o d c o r r e l a t i o n b e t w e e n t h e s e n s i t i v i t y o f cells to V C R a n d A d r i a m y c i n . T h i s i n d i c a t e s t h a t t h e m u l t i d r u g r e s i s t a n c e is e x p r e s s e d in t h e cell l i n e s t h a t s h o w e d l o w s e n s i t i v i t y to t h e d r u g s . T h e l e v e l s o f m u l t i d r u g r e s i s t a n c e a m o n g t h e 8 cell lines in v i t r o w e r e as f o l l o w s ; S H I N - 3 ( h i g h l y r e s i s t a n t g r o u p )
S. Sekiya et at.
82
100
SHIN-3
HAG-2
50-
L_
I00-
Hoe-21
50-
m
~"
t__
lOO
HOC~I
50-
100
HMOA
HUOCA-]
'1 i i
030.5 I
5 &l 0.5 1 Concentration
lO0
50-
100-
50-
I00-
50-
Fig. L Sensitivity of 8 h u m a n ovarian adenocarcinoma cell lines to vincristine in vitro. See Materials and Methods
~ ~ ~
-3
(/tg/m~,)
HUOA
\ Hoe-!
-2
A
100"
50-~-3
HOC-21
Fig. 2. Sensitivity of 8 h u m a n ovarian adenocarcinoma cell lines to adriamycin in vitro
o.i o'.5 i
5 o.~o.s C o n c e n t r a t i o n (pg/m~.)
Multidrug resistance in ovarian carcinoma
83
Table 2. Levels of P-glycoprotein and its mRNA and DNA amplification in 8 human ovarian adenocarcinoma cell lines Cell line
IC50~ Vincristine (~tg/ml)
Adriamycin (p.g/mt)
CMK ~ K 562~
NT~ NT
NT NT
SHIN-3 OMC-3 HUOA HMOA HAC-2 HOC-21 HOC-I HUOCA-II
>5 1.3 1.1 0.2 0.2 0.1 --, non-treated; - - e - - - , treated) or adriamycin (--A--, non-treated; - - A - - treated) on the growth of tumors developed by inoculation of 3 human ovarian adenocarcinoma cell lines in vivo. I indicates the death of nude mouse. See Materials and Methods
Weeks after inoculation
1
4.5kb
2
34
5
6
7 8 9
--
Fig. 4. Mdr 1 RNA expression in 8 human ovariand adenocarcinoma cell lines by a Northern blot. Lane 1, HeLa (negative control for mdr 1 RNA transcripts); Lane 2, HOC-21; Lane 3, SHIN-3 (positive) ; Lane 4, HUOA; Lane 5, OMC-3; Lane 6, HMOA; Lane 7, HOC-I; Lane 8, HAC-2; Lane 9, HUOCA-II. kb, kilobase
Multidrug resistance in ovarian carcinoma
85
Discussion
Detection of P-glycoprotein in ovarian carcinoma was first reported by Bell et al. [10] in 1985. A monoclonal antibody against P-glycoprotein was used to examine tumor samples from 5 patients with advanced ovarian carcinoma. High levels of P-glycoprotein (a molecular weight 170 kilodalton membrane component) were detected by using an immunoblotting method in samples from 2 patients who had undergone intensive chemotherapy. Bourhis et al. [11] analyzed mdr 1 gene expression in fresh ovarian carcinoma specimens from 50 patients by Northern and slot blot hybridization techniques using a mdr 1 cDNA probe. Mdr 1 transcripts, possibly the same ones observed in the present study, were detected in RNA preparations obtained from 8 of 10 patients who had been treated with VCR and/or Adriamycin. In contrast to these reports, Volm et al. [12] recently reported that the P-glycoprotein could be detected in ovarian carcinomas not previously treated with chemotherapy (one positive of 5 patients not previously treated). In parallel with these studies on human ovarian carcinoma, isolation of drugresistant derivatives from drug-sensitive parent human ovarian carcinoma cell lines by continuous exposure to increasing concentrations of VCR in vitro has been reported [13, 14]. The acquired multidrug resistant derivates showed extremely high ranges in resistance (more than 1,000-fold) and demonstrated corresponding overexpression of P-glycoprotein at both protein and mRNA levels. As compared with these in vitro models, range in resistance appeared to be low in our human ovarian carcinoma cell lines and the levels to be similar to those in ovarian carcinoma specimens. In our present results, only one (SHIN3) of 8 cell lines that appeared to be multidrug resistant in vitro and in vivo showed mdr 1 transcripts with a slight increase of P-glycoprotein. No cell lines showed the amplification of mdr 1 DNA. Bradley et al. [15] also reported an overexpression of mdr 1 gene with no DNA amplification in a multidrugresistant human ovarian carcinoma cell line. This weak multidrug resistance process appears to be characteristics of ovarian carcinoma and gene expression of the multidrug resistance phenotype is probably under complex control in ovarian carcinoma. Acknowledgement~ We thank Dr. T. Sato, Department of Pediatrics, Chiba University School of Medicine, for the gift of the CMK cell line.
References I. Gerlach J, Kartner N, Bell DR, Ling V (1986) Multidrug resistance. Cancer Surv 5:25-46 2. Gottesman MM, Pastan I (1988) Resistance to multiple chemotherapeutic agents in human cancer cells. TIPS 9:54-58 3. Tsuruo T (1988) Mechanisms of multidrug resistance and implications for therapy. Jpn J Cancer Res (Gann) 79:285-296 4. Sekiya S, Tanaka N, Takamizawa H (1990) Production of various marker substances in human ovarian cancer cell lines of epithelial origin. Arch Gynecol Obstet 248:103-110
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