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In vivo cisplatin resistance of rat ascites hepatoma AH66
CANCER LETTERS
ELSEVIER
Cancer
Letters
108 (1996)
1.53-156
In vivo cisplatin resistance of rat ascites hepatoma AH66 Tomoyoshi Minaminoa, Masaaki Nomurab, Mitsuo Tamaic, Shuzo Moritanid, Tohru Ohshima”, Ken-ichi Miyamoto b,ct* “Department of Legal Medicine, Kanazawa University School of Medicine, 13-1 Takara-machi, Kanazawa 920, Japan bFaculty of Pharmaceutical Sciences, Hokuriku University, Ho-3 Kanagawa-machi, Kanazawa 920-l I, Japan cDepartment of Pharmacology and Pharmaceutics, Division of Pharmacy, and Health Sciences, Graduate School of Pharmaceutical Kanazawa University, 13-I Takara-machi, Kanazawa 920, Japan ‘Fukui Prefectural University College of Nursing, 4-1 -I Kenjojima, Matsuoka-cho, Fukui 910-I 1, Japan Received
16 May
1996; revision
received
3 July 1996; accepted
Sciences,
4 July 1996
Abstract The rat ascites hepatoma AH66 cell line had higher resistance to cisplatin than a variant AH66F cell line in in vivo experiments, though in vitro sensitivities to cisplatin of both cell lines were similar in a medium containing 5% fetal calf serum (FCS). WhenAH cellswereculturedin the mediumcontaining5% ascitesfluid (ASF), the sensitivityof AH66 cellsto
cisplatinwassignificantlylower thanthat in the mediumcontaining5% FCS,but the sensitivityof AH66F cellswasalmost same in the case of 5% FCS. The metallothionein
(MT) contents in AH66 cells, but not in AH66F cells, increased with the
incubationtime in the mediumcontainingASF. MT in AH66 cells wasalsoinducedby treatmentwith zinc ion, but the inductionin AH66F cellswasvery low. Theseresultsindicatethat AH66 cellshavea high ability to induceMT andthereby may acquirein vivo resistance to cisplatin. Keywords:
Rat ascites hepatoma; AH66 cells; Cisplatin; In vivo resistance; Ascites fluid; Metallothionein
1. Introduction The AH66 cell line is a rat asciteshepatoma cell line induced by dimethylaminoazobenzeneand established as a transplantable tumor [l]. We have previously reported that this cell line is highly resistant to several antitumor drugs, becauseof overexpression of P-glycoprotein [2-41 and an improved detoxifica-
* Corresponding
author.
0304-3835/96/$12.00 0 1996 Elsevier PII SO304-3835(96)04385-6
Science Ireland
tion system through glutathione (GSH) and glutathione S-transferase (GST) placental form [5], comparedwith the drug-sensitive variant AH66F. There are many reports on the relationships between cisplatin sensitivity and metallothionein (MT) contents and GST activities in tumor cells [610]. Although AH66 cells have more GSH and higher GST activity than AH66F cells, the in vitro sensitivity of AH66 cells to cisplatin was similar to that of AH66F cells [5,11]. However, we found that the therapeutic effect of cisplatin on AH6Bbearing rats was markedly less than on AH66F-bearing rats. In this
Ltd. All rights reserved
study, we investigated the cause of this in viva resrstance of AH66 cells to cisplatin.
2. Materials
rats at 37°C for 4X h m a CO? incubator. There was no difference between the cell growth in 5% FCS medium and 5% ASF medium. Cells were counted under a microscope, and the effects of cisplatin were expressed in term of the 5W growth-inhibitory concentration UC>,, i.
and methods
The AH66 and AH66F cell lines were supplied by the Department of Experimental Therapeutics, Cancer Research Institute. Kanazawa University. Cells were maintained by intraperitoneal (i.p.) passage in female Donryu rats weighing ltX- 150 g (Nippon SLC. Himamatsu) and harvested from the tumor-bearing rats 6- 10 days after the tumor transplantation. -’ .-.’
117 r,iw
Cells were suspended in 0.25 M sucrose and homogenized by sonication, and the homogenates were used for measurement of metallothionein (MT’) content by the cadmium-heme method (121. A rnixture consisting of a portion of the cell homogenate. 1 pg/ ml cadmium chloride. and SO mM Tris-I-ICI buffer (pH X.0) was incubated for 10 min at room temperature, then 0. I ml of 2% bovine hemoglobin was added and the mixture was incubated for 2 min more in hoiliug water, then centrifuged at 10 000 Y s for 5 min. Thk course was repeated three times. The cad.mium content in the supernatant was analyzed by hamelexs atomic absorption spectrophotometry, and the MT content was calculated.
thmlp?’
Cells (2 x IO’) were i.p. inoculated into ten rats in a group on Day I), and cisplatin was i.p. injected on Days I. 5. and 9. The effects of cisplatin were evaluated by the number of survivors for 60 days. and the percentage increase in the life span (o/OILS) calculated from the following equation: %ILS = [(T-C)/ C] x 100. where T and C represent the mean survival days of the treated group excluding regressors at 60 days and the mean survival days of the vehicle control group. respectively.
3. Results and discussion Table 1 shows the in vivo effects of cisplatin on AH-bearing rats. The life span of AH66F-bearing rats was prolonged by injection of cjsplatin in a dosedependent manner. and rats that were administered 7.0 mg/kg of cisplatin all survived for 60 days. On the other hand, the effect of cisplatin on AH66bearrtrg rats was small. while the in vitro sensitivity to cisplatin of AH66 cells was similar to that oi 4H66F cells when assayed in medium containing
Cells (5 x 105/ml) were cultured in RPM1 1640 medium with 109% fetal calf serum (FCS’I for 24 h and further cultured with or without cisplatin (Sigma Chemical Co.. St. Louis, MO) in medium with 5% FCS or 5% ascites fluid (ASF) from tumor-bearing lablc
1
In viva el’i’ects ot cisplatin DOW
on AH-beanng
rats
---.-__--
AH66 --..
~Illg/kkgl
“The survival
9.5 13.4 16.0 20.5 >60
--.--
* 3.9 iT 6.4 f 7.7 Ir 9.3
W-da! surviwrk
--
_---_I
.--l.l___ excluding
Survival days (Mean k SD)
ij jt L h IO
41.i 6X.4 11q.x
time and SOILS were calculated
---..
-.
QILS
Survival days” (Mean + SD) 0 Il.25 r1.s I .o 2.0 --.
-
AH66F
ho-day
-.---
-_.--
sur~iw~r~.
13.0 I 1.8 14.2 15.i 1 7.7 .._^...__ ----.
f 3.x _L 3.0 5 l.i - 7i ? (,,tj .-...----__..--
‘CILS
-9.’ ‘2.2 IT.ih.2 _.____.-__
_--
-_..._ N-day .survivor~
1, ti 0 ii ‘, -_ _...--_
T. Minamino
et al. I Cancer
Table 2
Letters
108 (1996)
153-156
0-
In vitro effects of ascites fluid on cisplatin-sensitivities
Cells
of AH cells
Go (PM) 5% FCS
5% ASP
AH66F
1.56 + 0.21
AH66
1.14 f 0.54
1.25 (1.42 3.98 (3.14
f 0.39 f 0.54)b f 0.78* + 0.43*)
Each value is the mean f SE of at least three experimet&From AH66-bearing rats.bValue in parenthesis is ICss for cisplatin in the medium containing 5% ascites fluid from AH66Fhearing rats.*Significantly different from the 5% fetal calf serum at P < 0.05. 1OiI
5% Arzk Z FCS (Table 2). We then examined in vitro sensitivities to cisplatin of AH cells in the medium containing ASF. The sensitivity of AH66F cells was not changed in the medium containing ASF, while that of AH66 cells was significantly less in ASF, from both AH66- and AH66F-bearing rats, than in FCS (Table 2). Consequently, the difference in the in vivo sensitivities between these cells seems to be based on the difference in responsiveness of the cells to some factors in ASF. Fig. 1 shows the changes of MT contents in AH cells when cells were cultured in 5% FCS or 5% 700
1
.
WO-
g wog L 400% =
300200100-l
) -24
I 0 Incubation
I 24
I 40
time(h)
Fig. 1. Changes of MT contents in AH cells during culture in 5% FCS or 5% ASF. AH66 cells (circles) and AH66F cells (triangles), which were harvested from the peritoneal cavity of the respective tumor-bearing rats, were cultured in medium containing 5% FCS for 24 h, then cultured in 5% FCS (open symbols) or 5% ASF (closed symbols) for 48 h. *Significantly different from the culture in FCS at P < 0.05.
Concentration
200 cl
ZnClp@M)
Fig. 2. Induction of MT in AH66 cells (circles) and AH66F cells (triangles) after exposure to ZnCl?. Cells were incubated in medium containing 5% FCS in the presence of the indicated concentrations of ZnClz for 24 h.
ASF. The MT content in AH66 cells just after harvest from the tumor-bearing rats was about three-fold higher than that in AH66F cells, as previously reported [ 111, but the MT level in AH66 cells was decreased to the level in AH66F cells by incubating them in the medium containing 5% FCS for 24 h, then changing the medium to 5% ASF. The MT content in AH66 cells increased with incubation time, but not in AH66F cells. It is known that in several types of cells, cellular MT is induced by zinc ion [13]. As shown in Fig. 2, the MT content in AH66 cells was also increased greatly by ZnC12 in a concentration-dependent manner up to 200 PM, but the induction in AH66F cells was very low. These results indicate that AH66 cells have a high ability to induce MT and thereby may acquire cisplatin resistance. ASF did not contain a detectable amount of zinc ion, but ASF should contain numerous kinds and amount of cytokines, and cytokines such as IL-10 and TNF-a have been shown to induce MT 114,151. Recently, it has been reported that there is a close relationship between reversing cisplatin resistance and induction -’ GST-a by IL-6 in human renal carcinoma [16]. However, although AH66 cells have high GST activity and much GSH, their sensitivity to cisplatin was not influenced by a GSH biosynthesis inhibitor, buthionine sulfoximine, and a GST-P selective substrate, ethacrynic acid [ll]. Therefore, the
cytotoxicity of cisplatin seems not to be modulated through the GSH/GST system. This study indicates that AH66 cells responsive to inducers of MT showed in vivo resistance to cisplatin. Although the molecular species inducing of MT in ASF must be identified, the in vivo cisplatin resistance of tumor cells should be considered in the clinic. Acknowledgements This work was supported in part by the Special Research Fund of Hokuriku University and by a Grant in Aid for Cancer Research from the Ministry of Health and Welfare of Japan. References Yoshida. T. (lYS6) Contributions of the ascites hepatoma 10 the concept of malignancy of cancer. Ann. N. Y. Acad. Sci.. 63, X52-881. Miyamoto, K.. Wakusawa, S. and Nakamura, S. (1992)Drug resistance dependent on different molecular size P-glycoproteins in Yoshida rat ascites hepatoma cells. Biochem. Phamdcc~i.,
43,
! 143-l
145.
Miyamoto, K., Wakusawa. S., Nakamura, S., Tajima. K. and Hidaka. H. ( 1992) Multidrug resistance in Yoshida rat ascites hepatoma cell lines, Anticancer Res.. 12. 649-654. Wakusawa. S.. Nakamura. S.. Inoko, K. and Miyamoto. K. (1992) Sensitivity to antitumor drugs and vinblastine binding to membrane in rat ascites hepatoma AH66 ceils. Chem. Pharm. Bull.. 40. 21X2-2184. Miyamoto. K.. Wakabayashi. D.. Minamino, 1‘. and Nomura. M. ( 1994) Glutathione-S-transferase P-form dependent chlorambucil resistance in Yoshida rat ascites hepatoma celt line\. Cancer Lett.. 78. 77-83. Hospers. G.A.P.. Mulder. N.H.. DeJong. B., DeLrvy, L... Uges. D.R.A.. Fichtinger-Schepman. A.M.J.. Scheper, R.J. and DeVries. E.G.E. (1988) Characterization of a human small cell lung carcinoma cell line with acquired resistance to cis-diamminedichloroplatinum(Ii) in vivo.Cancer Res.. 4X 6803-6807.
Kelly., S.L.. Ucisu. 4.. Irichcr. B., Hacker. M.P.. Hsrmcr. Li. and Laso, J.S i 19X8) Overexpression of metallotiionein> confers resistance to anticancer drugs, Science. 131, 18 131x1s” Sahurl. ‘I Sagawa. M.. One. M., Sakai. kl.. Muramalsu. LI. Kohno. K, and Ktrwano. M. (1989) Increased expression oi :lutathionc S-transferax gene in cis-diamminedichloroplati inurn(resistam variants of Chinese hamster. Cancer Reh.. 19. 7020-7025 I’cicher. B.A.. Holden. 5.A.. Wermdn. S.‘I ., &&mayor. 1t.A. Kanderkar. V., Rosbe. K.W.. Brann. T.W.. Korhut. 1‘:~. ;I& Frei 111. E. ( 100 I i Charactcristica of tive human tumor xii line:, and sublines resistant to cis-diamminedichkmplatinum(fI), int. J. Cancer. 17, 252-260. Kasahara. K.. Fujiwam. Y., Nishio. K., Ohmori. 1. Sugimoto. 1. Komiya, ti.. Mats&a. T. and Sai,jyo. N I 1991 I Metallothiunein content correlates with the \ensitivii;
ELSEVIER
Cancer
Letters
108 (1996)
1.53-156
In vivo cisplatin resistance of rat ascites hepatoma AH66 Tomoyoshi Minaminoa, Masaaki Nomurab, Mitsuo Tamaic, Shuzo Moritanid, Tohru Ohshima”, Ken-ichi Miyamoto b,ct* “Department of Legal Medicine, Kanazawa University School of Medicine, 13-1 Takara-machi, Kanazawa 920, Japan bFaculty of Pharmaceutical Sciences, Hokuriku University, Ho-3 Kanagawa-machi, Kanazawa 920-l I, Japan cDepartment of Pharmacology and Pharmaceutics, Division of Pharmacy, and Health Sciences, Graduate School of Pharmaceutical Kanazawa University, 13-I Takara-machi, Kanazawa 920, Japan ‘Fukui Prefectural University College of Nursing, 4-1 -I Kenjojima, Matsuoka-cho, Fukui 910-I 1, Japan Received
16 May
1996; revision
received
3 July 1996; accepted
Sciences,
4 July 1996
Abstract The rat ascites hepatoma AH66 cell line had higher resistance to cisplatin than a variant AH66F cell line in in vivo experiments, though in vitro sensitivities to cisplatin of both cell lines were similar in a medium containing 5% fetal calf serum (FCS). WhenAH cellswereculturedin the mediumcontaining5% ascitesfluid (ASF), the sensitivityof AH66 cellsto
cisplatinwassignificantlylower thanthat in the mediumcontaining5% FCS,but the sensitivityof AH66F cellswasalmost same in the case of 5% FCS. The metallothionein
(MT) contents in AH66 cells, but not in AH66F cells, increased with the
incubationtime in the mediumcontainingASF. MT in AH66 cells wasalsoinducedby treatmentwith zinc ion, but the inductionin AH66F cellswasvery low. Theseresultsindicatethat AH66 cellshavea high ability to induceMT andthereby may acquirein vivo resistance to cisplatin. Keywords:
Rat ascites hepatoma; AH66 cells; Cisplatin; In vivo resistance; Ascites fluid; Metallothionein
1. Introduction The AH66 cell line is a rat asciteshepatoma cell line induced by dimethylaminoazobenzeneand established as a transplantable tumor [l]. We have previously reported that this cell line is highly resistant to several antitumor drugs, becauseof overexpression of P-glycoprotein [2-41 and an improved detoxifica-
* Corresponding
author.
0304-3835/96/$12.00 0 1996 Elsevier PII SO304-3835(96)04385-6
Science Ireland
tion system through glutathione (GSH) and glutathione S-transferase (GST) placental form [5], comparedwith the drug-sensitive variant AH66F. There are many reports on the relationships between cisplatin sensitivity and metallothionein (MT) contents and GST activities in tumor cells [610]. Although AH66 cells have more GSH and higher GST activity than AH66F cells, the in vitro sensitivity of AH66 cells to cisplatin was similar to that of AH66F cells [5,11]. However, we found that the therapeutic effect of cisplatin on AH6Bbearing rats was markedly less than on AH66F-bearing rats. In this
Ltd. All rights reserved
study, we investigated the cause of this in viva resrstance of AH66 cells to cisplatin.
2. Materials
rats at 37°C for 4X h m a CO? incubator. There was no difference between the cell growth in 5% FCS medium and 5% ASF medium. Cells were counted under a microscope, and the effects of cisplatin were expressed in term of the 5W growth-inhibitory concentration UC>,, i.
and methods
The AH66 and AH66F cell lines were supplied by the Department of Experimental Therapeutics, Cancer Research Institute. Kanazawa University. Cells were maintained by intraperitoneal (i.p.) passage in female Donryu rats weighing ltX- 150 g (Nippon SLC. Himamatsu) and harvested from the tumor-bearing rats 6- 10 days after the tumor transplantation. -’ .-.’
117 r,iw
Cells were suspended in 0.25 M sucrose and homogenized by sonication, and the homogenates were used for measurement of metallothionein (MT’) content by the cadmium-heme method (121. A rnixture consisting of a portion of the cell homogenate. 1 pg/ ml cadmium chloride. and SO mM Tris-I-ICI buffer (pH X.0) was incubated for 10 min at room temperature, then 0. I ml of 2% bovine hemoglobin was added and the mixture was incubated for 2 min more in hoiliug water, then centrifuged at 10 000 Y s for 5 min. Thk course was repeated three times. The cad.mium content in the supernatant was analyzed by hamelexs atomic absorption spectrophotometry, and the MT content was calculated.
thmlp?’
Cells (2 x IO’) were i.p. inoculated into ten rats in a group on Day I), and cisplatin was i.p. injected on Days I. 5. and 9. The effects of cisplatin were evaluated by the number of survivors for 60 days. and the percentage increase in the life span (o/OILS) calculated from the following equation: %ILS = [(T-C)/ C] x 100. where T and C represent the mean survival days of the treated group excluding regressors at 60 days and the mean survival days of the vehicle control group. respectively.
3. Results and discussion Table 1 shows the in vivo effects of cisplatin on AH-bearing rats. The life span of AH66F-bearing rats was prolonged by injection of cjsplatin in a dosedependent manner. and rats that were administered 7.0 mg/kg of cisplatin all survived for 60 days. On the other hand, the effect of cisplatin on AH66bearrtrg rats was small. while the in vitro sensitivity to cisplatin of AH66 cells was similar to that oi 4H66F cells when assayed in medium containing
Cells (5 x 105/ml) were cultured in RPM1 1640 medium with 109% fetal calf serum (FCS’I for 24 h and further cultured with or without cisplatin (Sigma Chemical Co.. St. Louis, MO) in medium with 5% FCS or 5% ascites fluid (ASF) from tumor-bearing lablc
1
In viva el’i’ects ot cisplatin DOW
on AH-beanng
rats
---.-__--
AH66 --..
~Illg/kkgl
“The survival
9.5 13.4 16.0 20.5 >60
--.--
* 3.9 iT 6.4 f 7.7 Ir 9.3
W-da! surviwrk
--
_---_I
.--l.l___ excluding
Survival days (Mean k SD)
ij jt L h IO
41.i 6X.4 11q.x
time and SOILS were calculated
---..
-.
QILS
Survival days” (Mean + SD) 0 Il.25 r1.s I .o 2.0 --.
-
AH66F
ho-day
-.---
-_.--
sur~iw~r~.
13.0 I 1.8 14.2 15.i 1 7.7 .._^...__ ----.
f 3.x _L 3.0 5 l.i - 7i ? (,,tj .-...----__..--
‘CILS
-9.’ ‘2.2 IT.ih.2 _.____.-__
_--
-_..._ N-day .survivor~
1, ti 0 ii ‘, -_ _...--_
T. Minamino
et al. I Cancer
Table 2
Letters
108 (1996)
153-156
0-
In vitro effects of ascites fluid on cisplatin-sensitivities
Cells
of AH cells
Go (PM) 5% FCS
5% ASP
AH66F
1.56 + 0.21
AH66
1.14 f 0.54
1.25 (1.42 3.98 (3.14
f 0.39 f 0.54)b f 0.78* + 0.43*)
Each value is the mean f SE of at least three experimet&From AH66-bearing rats.bValue in parenthesis is ICss for cisplatin in the medium containing 5% ascites fluid from AH66Fhearing rats.*Significantly different from the 5% fetal calf serum at P < 0.05. 1OiI
5% Arzk Z FCS (Table 2). We then examined in vitro sensitivities to cisplatin of AH cells in the medium containing ASF. The sensitivity of AH66F cells was not changed in the medium containing ASF, while that of AH66 cells was significantly less in ASF, from both AH66- and AH66F-bearing rats, than in FCS (Table 2). Consequently, the difference in the in vivo sensitivities between these cells seems to be based on the difference in responsiveness of the cells to some factors in ASF. Fig. 1 shows the changes of MT contents in AH cells when cells were cultured in 5% FCS or 5% 700
1
.
WO-
g wog L 400% =
300200100-l
) -24
I 0 Incubation
I 24
I 40
time(h)
Fig. 1. Changes of MT contents in AH cells during culture in 5% FCS or 5% ASF. AH66 cells (circles) and AH66F cells (triangles), which were harvested from the peritoneal cavity of the respective tumor-bearing rats, were cultured in medium containing 5% FCS for 24 h, then cultured in 5% FCS (open symbols) or 5% ASF (closed symbols) for 48 h. *Significantly different from the culture in FCS at P < 0.05.
Concentration
200 cl
ZnClp@M)
Fig. 2. Induction of MT in AH66 cells (circles) and AH66F cells (triangles) after exposure to ZnCl?. Cells were incubated in medium containing 5% FCS in the presence of the indicated concentrations of ZnClz for 24 h.
ASF. The MT content in AH66 cells just after harvest from the tumor-bearing rats was about three-fold higher than that in AH66F cells, as previously reported [ 111, but the MT level in AH66 cells was decreased to the level in AH66F cells by incubating them in the medium containing 5% FCS for 24 h, then changing the medium to 5% ASF. The MT content in AH66 cells increased with incubation time, but not in AH66F cells. It is known that in several types of cells, cellular MT is induced by zinc ion [13]. As shown in Fig. 2, the MT content in AH66 cells was also increased greatly by ZnC12 in a concentration-dependent manner up to 200 PM, but the induction in AH66F cells was very low. These results indicate that AH66 cells have a high ability to induce MT and thereby may acquire cisplatin resistance. ASF did not contain a detectable amount of zinc ion, but ASF should contain numerous kinds and amount of cytokines, and cytokines such as IL-10 and TNF-a have been shown to induce MT 114,151. Recently, it has been reported that there is a close relationship between reversing cisplatin resistance and induction -’ GST-a by IL-6 in human renal carcinoma [16]. However, although AH66 cells have high GST activity and much GSH, their sensitivity to cisplatin was not influenced by a GSH biosynthesis inhibitor, buthionine sulfoximine, and a GST-P selective substrate, ethacrynic acid [ll]. Therefore, the
cytotoxicity of cisplatin seems not to be modulated through the GSH/GST system. This study indicates that AH66 cells responsive to inducers of MT showed in vivo resistance to cisplatin. Although the molecular species inducing of MT in ASF must be identified, the in vivo cisplatin resistance of tumor cells should be considered in the clinic. Acknowledgements This work was supported in part by the Special Research Fund of Hokuriku University and by a Grant in Aid for Cancer Research from the Ministry of Health and Welfare of Japan. References Yoshida. T. (lYS6) Contributions of the ascites hepatoma 10 the concept of malignancy of cancer. Ann. N. Y. Acad. Sci.. 63, X52-881. Miyamoto, K.. Wakusawa, S. and Nakamura, S. (1992)Drug resistance dependent on different molecular size P-glycoproteins in Yoshida rat ascites hepatoma cells. Biochem. Phamdcc~i.,
43,
! 143-l
145.
Miyamoto, K., Wakusawa. S., Nakamura, S., Tajima. K. and Hidaka. H. ( 1992) Multidrug resistance in Yoshida rat ascites hepatoma cell lines, Anticancer Res.. 12. 649-654. Wakusawa. S.. Nakamura. S.. Inoko, K. and Miyamoto. K. (1992) Sensitivity to antitumor drugs and vinblastine binding to membrane in rat ascites hepatoma AH66 ceils. Chem. Pharm. Bull.. 40. 21X2-2184. Miyamoto. K.. Wakabayashi. D.. Minamino, 1‘. and Nomura. M. ( 1994) Glutathione-S-transferase P-form dependent chlorambucil resistance in Yoshida rat ascites hepatoma celt line\. Cancer Lett.. 78. 77-83. Hospers. G.A.P.. Mulder. N.H.. DeJong. B., DeLrvy, L... Uges. D.R.A.. Fichtinger-Schepman. A.M.J.. Scheper, R.J. and DeVries. E.G.E. (1988) Characterization of a human small cell lung carcinoma cell line with acquired resistance to cis-diamminedichloroplatinum(Ii) in vivo.Cancer Res.. 4X 6803-6807.
Kelly., S.L.. Ucisu. 4.. Irichcr. B., Hacker. M.P.. Hsrmcr. Li. and Laso, J.S i 19X8) Overexpression of metallotiionein> confers resistance to anticancer drugs, Science. 131, 18 131x1s” Sahurl. ‘I Sagawa. M.. One. M., Sakai. kl.. Muramalsu. LI. Kohno. K, and Ktrwano. M. (1989) Increased expression oi :lutathionc S-transferax gene in cis-diamminedichloroplati inurn(resistam variants of Chinese hamster. Cancer Reh.. 19. 7020-7025 I’cicher. B.A.. Holden. 5.A.. Wermdn. S.‘I ., &&mayor. 1t.A. Kanderkar. V., Rosbe. K.W.. Brann. T.W.. Korhut. 1‘:~. ;I& Frei 111. E. ( 100 I i Charactcristica of tive human tumor xii line:, and sublines resistant to cis-diamminedichkmplatinum(fI), int. J. Cancer. 17, 252-260. Kasahara. K.. Fujiwam. Y., Nishio. K., Ohmori. 1. Sugimoto. 1. Komiya, ti.. Mats&a. T. and Sai,jyo. N I 1991 I Metallothiunein content correlates with the \ensitivii;