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Possible mechanism for differences in sensitivity to cis-platinum in human prostate tumor cell lines
163
Cancer Letters, 31 (1986) 163-169 Elsevier Scientific Publishers Ireland Ltd.
POSSIBLE MECHANISM FOR DIFFERENCES IN SENSITIVITY CIS-PLATINUM IN HUMAN PROSTATE TUMOR CELL LINES
TO
SYLVIA A. METCALFEa** , KELVIN CAINb and BRIDGET T. HILL”* * =Laboratory of Cellular Chemotherapy, Imperial Cancer Research Fund, Lincoln’s Inn Fields, London WCZA 3PX, and Institute of Urology, Shaftesbury Avenue, London WC2 and bToxicology Unit, MRC Laboratories, Woodmansterne Road, Carshalton, Surrey (U.K.) (Received 19 December 1985) (Revised version received 21 February 1986) (Accepted 20 March 1986)
SUMMARY
Two human prostate tumor cell lines which exhibit a 2.4-fold difference in sensitivity to cis-platinum (cis-Pt) were found to possess slightly different sulphydryl contents, and the presence of a metallothionein-like zinc-binding protein was demonstrated in the line exhibiting relative resistance to cis-Pt. Although these factors have been postulated to play a role in the mechanism(s) of resistance to cisBt in other cell types, preliminary data in this report suggest that differences found in drug uptake and subsequent binding to DNA are most likely responsible for variations in cis-Pt sensitivity displayed by these prostate tumor cell lines.
INTRODUCTION
Cis-platinum (cis:Pt) is one of the few clinically useful drugs in prostate cancer [ 61. Unfortunately, as a single agent, response rates range only from 12-31% suggesting that the majority of these tumors exhibit resistance to cis-Pt. Considerable evidence exists to suggest that cis-Pt exerts its cytotoxic effects via interactions with DNA (reviewed in Ref. 21), causing primarily DNA-DNA intra- and inter-strand cross-links and some DNA-protein crosslinks. The ability of cells to repair these DNA-platinum lesions has been implicated in the recovery of cells from cisPt cytotoxicity [ 19,211. However, various factors which might prevent cis-Pt from interacting with DNA could also be important including: reduced cellular uptake of drug or its associa*Present address: Cornell University Medical Center - New York Hospital, Department of Pediatric Hematology/Oncology, 525 East 68th Street, New York, NY 10021 (U.S.A.). **To whom all correspondence should be addressed. 0304-3835/86/$03.50 01986 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
164
tion with other nucleophiles prior to entering the nucleus, particularly with molecules containing sulphydryl (SH) moieties such as reduced glutathione (GSH) [3,17] and the cysteine-rich cytosolic protein, metallothionein (MT), which binds certain metals [ 1,271. Two cell lines derived from human prostate carcinomas, which are both more sensitive to cis-Pt in vitro compared with other human tumor cell lines [5,22], have, however, shown variable degrees of sensitivity to cis-Pt [20]: the DU145 cells were 2.4-fold less sensitive than PC3MA2 cells at the ID,,, level (i.e. drug concentration for a 24-h exposure causing 70% inhibition of cell survival in agarose). This present study describes results indicating possible mechanism(s) for differences in sensitivity to cis-Pt in these 2 human prostate tumor cell lines. MATERIALS
AND METHODS
Materials Cis-Pt was a gift from Bristol Meyers, UK and ‘9’-Pt-labeled drug was a generous gift from Dr. H.L. Sharma, University of Manchester, U.K. PC3MA2 cells, provided by Dr. M.E. Kaighn, Frederick Cancer Research Center, MD 21701, U.S.A., and DU145 cells, provided by Dr. J. Fogh, Sloan-Kettering Institute, Rye, NY 10580, U.S.A., were grown as monolayers in RPM1 1640 medium with 10% fetal calf serum and survival was assessed by colony formation [ 201. Methods GSH content (specifically, non-protein SH content) of cells was determined by the spectrophotometric method of Beutler et al. as modified by Johnson [ 121. Glutathione disulfide (GSSG) levels were estimated using the method of Suzukake et al. [25]. Metallothionein estimation in the cells was performed by gel filtration, ion-exchange chromatography and metal analysis using flame atomic absorption spectrophotometry [ 21. Cis-Pt uptake and binding studies used drug radiolabeled with ‘91Pt with a half-life of 3 days. To investigate macromolecular binding of the drug, cells (20 X 9 cm dishes) were treated with 0.9 Mg/ml(16.8 pCi/mg) drug for 2 h at 37°C (dose equivalent to IC90 for PC3MA2 and ID6,-,for DU145 for 24-h exposure) and washed. Cellular DNA and protein were isolated as described [ 19,231 and associated radioactivity was determined. DNA was assayed by the Giles and Myers method [ 81. In uptake studies, cells were treated with labeled drug (0.9 and 1.8 pg/ml) in serum-free medium over 4 h, washed and their radioactivity was determined. RESULTS
AND DISCUSSION
Endogenous GSH may be involved in interaction with cis-Pt [17] and alkylating agents [ 251, and binding of such electrophiles to nucleophilic SH-
165
containing compounds prior to interaction with DNA has been shown to reduce cytotoxicity in vivo [ll] and in vitro [24]. In addition buthionine sulfoximine mediated GSH depletion has been shown to augment cisplatin cytotoxicity in human ovarian carcinoma cell lines [9] . In this present study we examined levels of non-protein SH in 2 cell lines to see whether inherent differences existed which may be associated with their different susceptibilities to the cytotoxic effects of cis-Pt. A continuous decrease in SH content with time after plating was seen in PC3MA2 cells (Table l), similar to that observed in other lines [lo]. However, the SH content of DU145 cells was slightly elevated at day 2, the time when cells were exposed to drug in colony-forming assays used to determine their cis-Pt sensitivities. An increase in SH at this time has also been reported in a human lung tumor cell line [ 181. The total thiol content of DU145 cells was marginally greater (1.3-fold) than that of PC3MA2 cells at day 2, whilst the GSSG levels of the 2 lines were comparable at 0.3 nmol/lO’ cells. However, this difference is probably insufficient to account for the 2.4-fold resistance to cis-Pt demonstrated in these DU145 cells, as judged by a comparison of ID,, values obtained from clonogenic assay data [ 201. Although the GSH assay used in this study permits measurement of nonprotein SH, this method does not exclude determination of SH due to metallothionein (MT). This protein (6000 kDa) binds certain metals, and is considered to be involved in metabolism and storage of zinc and copper [ 271, and has been detected in mammalian prostate gland [26] which is also relatively rich in zinc [ 161. MT has also been found in cell lines, usually in response to cadmium treatment [ 141. Bakka and co-workers demonstrated resistance to cis-Pt [ 11, chlorambucil and radiation [ 61 in a cadmium-human normal cell line and a mouse fibroblast line, both of which had high MT contents. Recently it has been reported that MT is a major cellular site of binding of cis-Pt in Ehrlich cells in culture at growth inhibitory concentrations [ 151. The two cell lines used in this study were therefore examined for the presence of MT. Figure 1 shows gel filtration elution profiles of cytosol preparations as indicated by zinc distribution profiles. A central single zincTABLE 1 SULFYDRYL CONTENTS OF HUMAN PROSTATE MONOLAYER CULTURES Cell line
TUMOR CELLS GROWN AS
SH content (nmol/lOh cells)a Day 0
Day 2
Day 3
Day 9
PC3MA2
22.0 * 0.3
18.8 +_0.3
12.8 +_0.4
5.0 + 0.1
DU145
19.1 + 0.01
24.5 2 3.1
5.6% 1.6
3.3 + 0.1
aValues are the mean of at least 4 determinations
+_S.E.M.
.
DU145
o PC3MA2
t
Fraction No.
2Ve ho Fig. 1. Gel filtration elution profiles of cytosol from PC3MA2 and DU145 cell lines as described in text. Zinc levels were measured in the fractions by flame atomic absorption spectrophotometry. An elution volume of V’V, = 1 B-2 .O corresponds to the MT peak.
containing peak eluting at VJV,, = 1.8-2.0, strongly indicative of an MTlike protein [27], was detected in DU145 cells. Ion-exchange chromatography of the fractions eluting at this volume resolved this peak into 2 zincbinding components (data not shown) confirming the identity of the MT-like protein [ 27 J . Giles and Cousins [ 71 have shown the presence of an MT-like protein associated with 65Zn following treatment of PC3 prostate cells with the radioisotope, but in this present study the content of this protein in the PC3MA2 subline was negligible (Fig. 1). Preliminary studies were performed to evaluate the uptake of labeled cisPt and its covalent binding to macromolecules in the 2 cell lines. At a dose level of 0.9 Erg/ml,the uptake of ‘91Pt-labeled drug was 3.2-fold greater by 2 h (2120 cpm/lO’ PC3MA2 cells and 660 cpm/lO* DU145 cells) and 1.5fold greater by 4 h (5340 cpm/lO’ PC3MA2 cells and 1940 cpm/108 DU145 cells) in the more sensitive PC3MA2 cells. At 1.8 E.cg/ml,uptake was 2.8-fold greater by 2 h. These 2 dose levels represent the range of doses at which the 2 cell lines show 2- to 3-fold differences in sensitivity [ 51. The increased uptake was paralleled by an elevated level of binding (approx. 4-fold) to DNA of 19*Ptin PC3MA2 cells (Table 2). Binding to protein was also about
167 TABLE 2 DNA BINDING OF lQIPt FOLLOWING (16.8 rCi/mg) cis-Pt FOR 2 h AT 37°C Cell line
DU145
TO 0.9 r&ml
lglPt bound to DNA (cpm/lO’
PC3MA2
IN VITRO DRUG EXPOSURE
cells)
(cpmlg DNA)
3560
6.25 x 10’
844
1.62 x 10’
4-fold higher in this line (data not shown). Similar results have been observed from experiments repeated at least twice. An extended time course and dose response of both drug uptake and binding to DNA has not been possible because of the large numbers of cells required and the difficulty of working with this radiolabeled cisPt with its short half-life and low specific activity. Therefore differences in repair of platinum-induced lesions cannot be ruled out as an explanation of the differential cytotoxicities. These initial studies provide the first such reported data with human prostate tumor cell lines indicating that laboratory studies are now feasible using human tumor material. These preliminary results would indicate that in these two particular lines, the difference in uptake of cis-Pt appears the most likely mechanism to account for variations in subsequent macromolecular binding and therefore responsible for their different drug sensitivities. However, it is not possible to exclude involvement of MT in sequestering cis-Pt. Further experiments are required to determine the role of this protein in resistance to cis-Pt in prostate cancer. Work is in progress to develop a higher level of resistance to cis-Pt and examine any concomitant alteration in these parameters. Similarly, it should be possible to manipulate MT levels, either by developing resistance to cadmium [ 1 ] or by treatment with dexamethazone, a known inducer of MT in cell lines [14], and determine whether this has any effect on cis-Pt cytotoxicity. REFERENCES Bakka, A., Endresen, L., Johnsen, A.B.S., Edminson, P.D and Rugstad, H.E. (198 1) Resistance against cis-dichlorodiammineplatinum in cultured cells with a high content of metallothionein. Toxicol. Appl. Pharmacol., 61,215-226. Cain, K.and Skilleter, D.N. (1983) Comparison of cadmium-metallothionein synthesis in parenchymal and non-parenchymal rat liver cells. Biochem. J., 210, 764-773. Connors, T. (1966) Protection against the toxicity of alkylating agents by thiols: the mechanism of protection and its relevance to cancer chemotherapy. A review. Eur. J. Cancer, 2,293-305. DeWys, D. (1982) The management of disseminated prostatic cancer. In: Principles of Cancer Treatment, pp. 552-557. Editors: S.K. Carter, E. Glatstein and R.B. Livingston. McGraw Hill, New York.
168 5 Drewinko, B. and Gottlieb, J.A. (1975) Action of cis-dichlorodiammineplatinum (II) (NSC-119875) at the cellular level. Cancer Chemother. Rep., 59,665-673. 6 Endresen, L., Bakka, A. and Rug&ad, H.E. (1983) Increased resistance to chlorambucil in cultured cells with a high concentration of cytoplasmic metallothionein. Cancer Res., 43, 2918-2926. Giles, P.J. and Cousins, R.J. (1982) Hormonal regulation of zinc metabolism in a human prostatic carcinoma cell line (PC-3). Cancer Res., 42, 2-7. Giles, K.W. and Myers, A. (1965) An improved diphenylamine method for the estima, tion of deoxyribonucleic acid. Nature, 206, 93. Hamilton, T.C., Winker, M.A., Louie, K.G., Batist, G., Behrens, B.S., Tsuruo, T., Grotzinger, K.R., McKay, W.M., Young, R.C. and Ozols, R.F. (1985) Augmentation of adriamycin, melphalan, and cisplatin cytotoxicity in drug-resistant and -sensitive human ovarian carcinoma cell lines by buthionine sulfoximine mediated glutathione depletion. Biochem. Pharmacol., 34,2583-2586. 10 Horikawa, M. (1968) Biochemical and genetical studies on radioresistant mouse cells in culture. Jpn. J. Gen., 43, 349-364. 11 Ishizawa, M., Taniguchi, S. and Baba, T. (1981) Protection by sodium thiosulfate and thiourea against lethal toxicity of cis-dichlorodiammineplatinum (II) in bacteria and mice. Jpn. J. Pharmacol., 31,883-889. 12 Johnson, M.K. (1966) Preparation of deproteinised tissue extracts for chromatography and assay of compounds related to glutathione. J. Chromatogr., 23,474-475. 13 Karin, M. and Herschman, H.R. (1980) Glucocorticoid hormone receptor mediated induction of metallothionein synthesis in HeLa cells. J. Cell. Physiol., 103, 35-40. 14 Koch, J., Wielgus, S., Shankara, B., Saryan, L.A., Shaw, C.F. and Petering, D.H. (1980) Zinc-, copper- and cadmium-binding protein in Ehrlich ascites tumour cells. Biochem. J., 189,95-104. 15 Kraker, A., Schmidt, J., Krezoski, S. and Petering, D.H. (1985) Binding of cisdichlorodiammine platinum (II) to metallothionein in Ehrlich cells. Biochem. Biophys. Res. Commun., 130, 786-792. 16 Leissner, K.-H., Fjelkegard, B. and Tisell, L.-E. (1980) Concentration and content of zinc in the human prostate. Invest. Urol., 18, 32-35. 17 Litterst, C.L., Tong, S., Hirokata, Y. and Siddik, Z.H. (1982) Alterations in hepatic and renal levels of glutathione and activities in glutathione S-transferases from rats treated with cis-dichlorodiammineplatinum (II). Cancer Chemother. Pharmacol., 8, 67-71. 18 Post, G.B., Keller, D.A., Connor, K.A. and Menzel, D.B. (1983) Effects of culture conditions on glutathione content of A549 cells. Biochem. Biophys. Res. Commun., 114,737-742. 19 Pera, M.F., Rawlings, C.J. and Roberts, J.J. (1981) The role of DNA repair in the recovery of human cells from cisplatin toxicity. Chem.-Biol. Interact., 37, 245-261. 20 Metcalfe, S.A., Whelan, R.D.H., Masters, J.R.W. and Hill, B.T. (1983)Zn vitroresponses of human tumour cell lines to a range of antitumour agents. Int. J. Cancer, 32, 351358. 21 Roberts, J.J. and Pera, M.F. (1983) Action of platinum antitumour drugs. In: Molecular Aspects of Anticancer Drug Action, pp. 183-231. Editors: S. Neidle and M. Waring. Macmillan Press, London. 22 Roberts, J.J. and Thomson, A.J. (1979) The mechanism of action of antitumour platinum compounds. Prog. Nucleic Acid Res. Mol. Biol., 22, 71-133. 23 Roberts, J.J., Pascoe, J.M., Plant, J.E., Sturrock, J.E. and Crathorn, A.R. (1971) Quantitative aspects of the repair of alkylated DNA in cultured mammalian cells. I. The effect of HeLa and Chinese hamster cell survival of alkylation of cellular macromolecules. Chem.-Biol. Interact., 3, 29-47. 24 Shrieve, D.C. and Harris, J.W. (1982) Protection against cis-dichlorodiammine Pt(I1) cytotoxicity in vitro by cysteamine. Int. J. Radiat., 8, 585-588.
169 25 Suzukake, K., Petro, B.J. and Vistica, D.T. (1982) Retention in glutathione content of L-PAM resistant L1210 cellsconfers sensitivity. Biochem. Pharmacol., 31,121-124. 26 Waalkes, M.P., Donovan, M.P. and Thomas, J.A. (1982) Cadmium-induced prostate metallothionein in the rabbit. Prostate, 3, 23-25. 27 Webb, M. and Cain, K. (1982) Functions of metallothionein. Biochem. Pharmacol., 31.137-142.
Cancer Letters, 31 (1986) 163-169 Elsevier Scientific Publishers Ireland Ltd.
POSSIBLE MECHANISM FOR DIFFERENCES IN SENSITIVITY CIS-PLATINUM IN HUMAN PROSTATE TUMOR CELL LINES
TO
SYLVIA A. METCALFEa** , KELVIN CAINb and BRIDGET T. HILL”* * =Laboratory of Cellular Chemotherapy, Imperial Cancer Research Fund, Lincoln’s Inn Fields, London WCZA 3PX, and Institute of Urology, Shaftesbury Avenue, London WC2 and bToxicology Unit, MRC Laboratories, Woodmansterne Road, Carshalton, Surrey (U.K.) (Received 19 December 1985) (Revised version received 21 February 1986) (Accepted 20 March 1986)
SUMMARY
Two human prostate tumor cell lines which exhibit a 2.4-fold difference in sensitivity to cis-platinum (cis-Pt) were found to possess slightly different sulphydryl contents, and the presence of a metallothionein-like zinc-binding protein was demonstrated in the line exhibiting relative resistance to cis-Pt. Although these factors have been postulated to play a role in the mechanism(s) of resistance to cisBt in other cell types, preliminary data in this report suggest that differences found in drug uptake and subsequent binding to DNA are most likely responsible for variations in cis-Pt sensitivity displayed by these prostate tumor cell lines.
INTRODUCTION
Cis-platinum (cis:Pt) is one of the few clinically useful drugs in prostate cancer [ 61. Unfortunately, as a single agent, response rates range only from 12-31% suggesting that the majority of these tumors exhibit resistance to cis-Pt. Considerable evidence exists to suggest that cis-Pt exerts its cytotoxic effects via interactions with DNA (reviewed in Ref. 21), causing primarily DNA-DNA intra- and inter-strand cross-links and some DNA-protein crosslinks. The ability of cells to repair these DNA-platinum lesions has been implicated in the recovery of cells from cisPt cytotoxicity [ 19,211. However, various factors which might prevent cis-Pt from interacting with DNA could also be important including: reduced cellular uptake of drug or its associa*Present address: Cornell University Medical Center - New York Hospital, Department of Pediatric Hematology/Oncology, 525 East 68th Street, New York, NY 10021 (U.S.A.). **To whom all correspondence should be addressed. 0304-3835/86/$03.50 01986 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
164
tion with other nucleophiles prior to entering the nucleus, particularly with molecules containing sulphydryl (SH) moieties such as reduced glutathione (GSH) [3,17] and the cysteine-rich cytosolic protein, metallothionein (MT), which binds certain metals [ 1,271. Two cell lines derived from human prostate carcinomas, which are both more sensitive to cis-Pt in vitro compared with other human tumor cell lines [5,22], have, however, shown variable degrees of sensitivity to cis-Pt [20]: the DU145 cells were 2.4-fold less sensitive than PC3MA2 cells at the ID,,, level (i.e. drug concentration for a 24-h exposure causing 70% inhibition of cell survival in agarose). This present study describes results indicating possible mechanism(s) for differences in sensitivity to cis-Pt in these 2 human prostate tumor cell lines. MATERIALS
AND METHODS
Materials Cis-Pt was a gift from Bristol Meyers, UK and ‘9’-Pt-labeled drug was a generous gift from Dr. H.L. Sharma, University of Manchester, U.K. PC3MA2 cells, provided by Dr. M.E. Kaighn, Frederick Cancer Research Center, MD 21701, U.S.A., and DU145 cells, provided by Dr. J. Fogh, Sloan-Kettering Institute, Rye, NY 10580, U.S.A., were grown as monolayers in RPM1 1640 medium with 10% fetal calf serum and survival was assessed by colony formation [ 201. Methods GSH content (specifically, non-protein SH content) of cells was determined by the spectrophotometric method of Beutler et al. as modified by Johnson [ 121. Glutathione disulfide (GSSG) levels were estimated using the method of Suzukake et al. [25]. Metallothionein estimation in the cells was performed by gel filtration, ion-exchange chromatography and metal analysis using flame atomic absorption spectrophotometry [ 21. Cis-Pt uptake and binding studies used drug radiolabeled with ‘91Pt with a half-life of 3 days. To investigate macromolecular binding of the drug, cells (20 X 9 cm dishes) were treated with 0.9 Mg/ml(16.8 pCi/mg) drug for 2 h at 37°C (dose equivalent to IC90 for PC3MA2 and ID6,-,for DU145 for 24-h exposure) and washed. Cellular DNA and protein were isolated as described [ 19,231 and associated radioactivity was determined. DNA was assayed by the Giles and Myers method [ 81. In uptake studies, cells were treated with labeled drug (0.9 and 1.8 pg/ml) in serum-free medium over 4 h, washed and their radioactivity was determined. RESULTS
AND DISCUSSION
Endogenous GSH may be involved in interaction with cis-Pt [17] and alkylating agents [ 251, and binding of such electrophiles to nucleophilic SH-
165
containing compounds prior to interaction with DNA has been shown to reduce cytotoxicity in vivo [ll] and in vitro [24]. In addition buthionine sulfoximine mediated GSH depletion has been shown to augment cisplatin cytotoxicity in human ovarian carcinoma cell lines [9] . In this present study we examined levels of non-protein SH in 2 cell lines to see whether inherent differences existed which may be associated with their different susceptibilities to the cytotoxic effects of cis-Pt. A continuous decrease in SH content with time after plating was seen in PC3MA2 cells (Table l), similar to that observed in other lines [lo]. However, the SH content of DU145 cells was slightly elevated at day 2, the time when cells were exposed to drug in colony-forming assays used to determine their cis-Pt sensitivities. An increase in SH at this time has also been reported in a human lung tumor cell line [ 181. The total thiol content of DU145 cells was marginally greater (1.3-fold) than that of PC3MA2 cells at day 2, whilst the GSSG levels of the 2 lines were comparable at 0.3 nmol/lO’ cells. However, this difference is probably insufficient to account for the 2.4-fold resistance to cis-Pt demonstrated in these DU145 cells, as judged by a comparison of ID,, values obtained from clonogenic assay data [ 201. Although the GSH assay used in this study permits measurement of nonprotein SH, this method does not exclude determination of SH due to metallothionein (MT). This protein (6000 kDa) binds certain metals, and is considered to be involved in metabolism and storage of zinc and copper [ 271, and has been detected in mammalian prostate gland [26] which is also relatively rich in zinc [ 161. MT has also been found in cell lines, usually in response to cadmium treatment [ 141. Bakka and co-workers demonstrated resistance to cis-Pt [ 11, chlorambucil and radiation [ 61 in a cadmium-human normal cell line and a mouse fibroblast line, both of which had high MT contents. Recently it has been reported that MT is a major cellular site of binding of cis-Pt in Ehrlich cells in culture at growth inhibitory concentrations [ 151. The two cell lines used in this study were therefore examined for the presence of MT. Figure 1 shows gel filtration elution profiles of cytosol preparations as indicated by zinc distribution profiles. A central single zincTABLE 1 SULFYDRYL CONTENTS OF HUMAN PROSTATE MONOLAYER CULTURES Cell line
TUMOR CELLS GROWN AS
SH content (nmol/lOh cells)a Day 0
Day 2
Day 3
Day 9
PC3MA2
22.0 * 0.3
18.8 +_0.3
12.8 +_0.4
5.0 + 0.1
DU145
19.1 + 0.01
24.5 2 3.1
5.6% 1.6
3.3 + 0.1
aValues are the mean of at least 4 determinations
+_S.E.M.
.
DU145
o PC3MA2
t
Fraction No.
2Ve ho Fig. 1. Gel filtration elution profiles of cytosol from PC3MA2 and DU145 cell lines as described in text. Zinc levels were measured in the fractions by flame atomic absorption spectrophotometry. An elution volume of V’V, = 1 B-2 .O corresponds to the MT peak.
containing peak eluting at VJV,, = 1.8-2.0, strongly indicative of an MTlike protein [27], was detected in DU145 cells. Ion-exchange chromatography of the fractions eluting at this volume resolved this peak into 2 zincbinding components (data not shown) confirming the identity of the MT-like protein [ 27 J . Giles and Cousins [ 71 have shown the presence of an MT-like protein associated with 65Zn following treatment of PC3 prostate cells with the radioisotope, but in this present study the content of this protein in the PC3MA2 subline was negligible (Fig. 1). Preliminary studies were performed to evaluate the uptake of labeled cisPt and its covalent binding to macromolecules in the 2 cell lines. At a dose level of 0.9 Erg/ml,the uptake of ‘91Pt-labeled drug was 3.2-fold greater by 2 h (2120 cpm/lO’ PC3MA2 cells and 660 cpm/lO* DU145 cells) and 1.5fold greater by 4 h (5340 cpm/lO’ PC3MA2 cells and 1940 cpm/108 DU145 cells) in the more sensitive PC3MA2 cells. At 1.8 E.cg/ml,uptake was 2.8-fold greater by 2 h. These 2 dose levels represent the range of doses at which the 2 cell lines show 2- to 3-fold differences in sensitivity [ 51. The increased uptake was paralleled by an elevated level of binding (approx. 4-fold) to DNA of 19*Ptin PC3MA2 cells (Table 2). Binding to protein was also about
167 TABLE 2 DNA BINDING OF lQIPt FOLLOWING (16.8 rCi/mg) cis-Pt FOR 2 h AT 37°C Cell line
DU145
TO 0.9 r&ml
lglPt bound to DNA (cpm/lO’
PC3MA2
IN VITRO DRUG EXPOSURE
cells)
(cpmlg DNA)
3560
6.25 x 10’
844
1.62 x 10’
4-fold higher in this line (data not shown). Similar results have been observed from experiments repeated at least twice. An extended time course and dose response of both drug uptake and binding to DNA has not been possible because of the large numbers of cells required and the difficulty of working with this radiolabeled cisPt with its short half-life and low specific activity. Therefore differences in repair of platinum-induced lesions cannot be ruled out as an explanation of the differential cytotoxicities. These initial studies provide the first such reported data with human prostate tumor cell lines indicating that laboratory studies are now feasible using human tumor material. These preliminary results would indicate that in these two particular lines, the difference in uptake of cis-Pt appears the most likely mechanism to account for variations in subsequent macromolecular binding and therefore responsible for their different drug sensitivities. However, it is not possible to exclude involvement of MT in sequestering cis-Pt. Further experiments are required to determine the role of this protein in resistance to cis-Pt in prostate cancer. Work is in progress to develop a higher level of resistance to cis-Pt and examine any concomitant alteration in these parameters. Similarly, it should be possible to manipulate MT levels, either by developing resistance to cadmium [ 1 ] or by treatment with dexamethazone, a known inducer of MT in cell lines [14], and determine whether this has any effect on cis-Pt cytotoxicity. REFERENCES Bakka, A., Endresen, L., Johnsen, A.B.S., Edminson, P.D and Rugstad, H.E. (198 1) Resistance against cis-dichlorodiammineplatinum in cultured cells with a high content of metallothionein. Toxicol. Appl. Pharmacol., 61,215-226. Cain, K.and Skilleter, D.N. (1983) Comparison of cadmium-metallothionein synthesis in parenchymal and non-parenchymal rat liver cells. Biochem. J., 210, 764-773. Connors, T. (1966) Protection against the toxicity of alkylating agents by thiols: the mechanism of protection and its relevance to cancer chemotherapy. A review. Eur. J. Cancer, 2,293-305. DeWys, D. (1982) The management of disseminated prostatic cancer. In: Principles of Cancer Treatment, pp. 552-557. Editors: S.K. Carter, E. Glatstein and R.B. Livingston. McGraw Hill, New York.
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