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[CANCER RESEARCH 47, 388-393, January 15, 1987] Characterization of a Human Squamous Carcinoma Cell Line Resistant to m-Diamminedichloroplatinum(II)1 Beverly A. Teicher,2 Sylvia A. Holden, Michael J. Kelley, Thomas C. Shea, Carol A. Cucchi, Andre Rosowsky, W. David Henner, and Emil Frei III Division of Cancer Pharmacology, Dana-Farber Cancer Institute, Boston, MA 02115 ABSTRACT We have developed a human head and neck squamous cell carcinoma cell line (SCC-25/CP) which is relatively stably resistant to m-diamminedichloroplatinum(II) (('1)1)1') after repeated exposure to escalating doses of the drug. The studies reported elucidate the mechanism(s) by which the SCC-25/CP cell line is resistant to CDDP. The SCC-25/CP cell line is approximately 30-fold resistant to CDDP, approximately 10fold resistant to carboplatin, and about 9-fold resistant to iproplatin. Using ["*"Pt]CDDP, we examined the levels of platinum in whole cells and cellular fractions of both the SCC-25 and SCC-25/CP cells after l h exposure to 100 iiM drug. The SCC-25 cells took up 30 pmol of platinum/ 10* cells in I h; 64% of the drug was in the nucleus and 21% in the cytosol. The SCC-25/CP cells took up 7 pmol of platinum/10' cells; of this, 41% was in the nucleus and 33% in the cytosol. The SCC-25 cell nuclei contained 331 pmol of platinum/mg protein and the cytosol 21 pmol of platinum/mg protein, whereas the SCC-25/CP cell nuclei con tained 47 pmol of platinum/mg protein and the cytosol 8.1 pmol/mg protein. The release of drug from both cell lines followed a very similar course and was most rapid over the first 6 h. There was no difference in the non-protein sulfhydryl content of the cell lines. The protein sulfhydryl content, as measured by Ellman's procedure, indicated that the SCC-25/CP cell line has approximately a 2-fold increase in protein sulfhydryl content compared to the SCC-25 cell line. The SCC-25/CP cell line is about 2-fold resistant to cadmium chloride at 50% cell kill and about 2.5-fold resistant at 1 log kill compared to the SCC-25 cell line. Glutathione transferase activity in crude cytoplasmic extracts was measured and found to be approximately 2- to 3fold higher in the CDDP resistant cells. The isoelectric point of the glutathione transferase isozyme was 4.8 in both the sensitive and resistant cell lines, suggesting induction of the predominant isozyme present in the parent cell line. By alkaline elution there was greater cross-link formation by CDDP in the SCC-25 cell line than in the SCC-25/CP cell line at the same drug concentrations. In conclusion, the mechanism of resistance of the SCC-25/CP cell line to CDDP is multifactorial, involving plasma membrane changes, increased cytosolic binding, and decreased DNA cross-linking. INTRODUCTION CDDP3 has demonstrated a broad range of activity against several malignancies in humans (1). CDDP is classified with the antitumor alkylating agents because it forms bidentate adducts with DNA (2-4). It is believed that DNA is the critical intracellular target of CDDP and that DNA cross-linking is the lethal lesion caused by this drug (2, 5, 6). Like many antineoplastic agents, CDDP is a potent mutagen, inducing frame shift and base substitution mutations in both bacterial and human cells (7-10). CDDP-sensitive and -resistant LI210 cell lines have been examined by several laboratories. These studies have Received 5/19/86; revised 9/12/86; accepted 10/14/86. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work is supported by National Cancer Institute grants 1RO1-CA36508, lPOl-CA38493,and 5F32-CA07821. 2To whom requests for reprints should be addressed. 3The abbreviations used are: CDDP, cú-diamminedichloroplatinum(II); GSH, reduced glutathione; GST, glutathione transferase; PAM, L-phenylalanine mus tard; FBS, fetal bovine serum; PBS, phosphate-buffered 0.9% saline solution; OPT, ortho-phthaldialdehyde; BSO, D,L-buthionine-S,A-sulfoxime. described changes in the plasma membrane (11-13) and changes at the level of DNA adduct formation (14) in the resistant cell lines. Experimental studies of other alkylating agents have indi cated that resistance to these drugs may occur by a variety of mechanisms. Goldenberg and Begleiter (IS) and Goldenberg et al. (16) found resistance to nitrogen mustard to be associated with a transport defect in the choline carrier impeding uptake of nitrogen mustard into the cells. Resistance to PAM has also been attributed to a transport defect, probably involving the leucine carrier (17, 18). In other studies, PAM resistance was associated with an elevation in intracellular glutathione (1922). Multifactorial resistance to PAM involving rate of drug efflux, sulfhydryl levels, and DNA interstrand cross-link for mation and repair has been reported in several cell lines (23, 24). Hilton and Colvin (25) have reported that cyclophosphamide-resistant human and rodent cell lines have increased levels of an aldehyde dehydrogenase that inactivates the aldophosphamide metabolite of the drug. Increased repair of DNA monoadducts via the action of guanine 06-methyltransferase has been described as the mechanism of resistance in l,3-bis(2chloroethyl)-l-nitrosourea-resistant bacterial and human cell lines (26-29). We have developed a human head and neck squamous cell carcinoma line (SCC-25/CP) which exhibits relatively stable resistance to cis-diannninedichloroplatinum(II) after repeated exposure to escalating doses of the drug (30). The studies reported here were designed to elucidate the mechanism(s) by which the SCC-25/CP cell line are resistant to CDDP. MATERIALS AND METHODS Drugs. CDDP, diammine [l,l-cyclobutanedicarboxylato(2)]-O,O'platinum(II) (carboplatin), and cw-dichloro-/ra/w-dihydroxobis(isopropylamine)platinum(IV) (iproplatin) were gifts from Johnson Matthey, Inc. (West Chester, PA). ["5mPt]cw-diamminedichloroplatinum(II) in isotonic saline was made available by Drs. J. D. I loes chele and F. F. Knapp, Jr. at Oak Ridge National Laboratories (Oak Ridge, TN) (31, 32). CdCl2 was purchased from Aldrich Chemical Co. (Milwaukee, WI). Cell Lines. SCC-25 and SCC-25/CP human squamous carcinoma of the head and neck cells grow as monolayers in Dulbecco-Vogt modified Eagle's minimum essential medium supplemented with antibiotics and 5% FBS (30). These cell lines have a plating efficiency of 10-30% and a doubling time of 48-50 h in vitro (33). For cloning, SCC-25 and SCC-25/CP cells were suspended by trypsinization, diluted in complete growth medium, and plated into 60- x 15-mm tissue culture dishes containing 5 ml of complete growth medium. Colonies grow to a countable size (>50 cells) in 2 weeks. The SCC-25/CP cell line has been maintained for 9 months in the absence of exposure to CDDP and the resistance of this cell line to CDDP has remained stable for that period. Survival Studies. SCC-25 and SCC-25/CP cells in exponential growth were exposed for 1 h to concentrations of CDDP, carboplatin, iproplatin, or CdCl2 ranging from 1-1000 /¿M in media without sera. The cells were then washed three times with PBS and plated for colony formation as described above. Each survival curve was determined in three independent experiments. 388 Downloaded from cancerres.aacrjournals.org on April 28, 2017. © 1987 American Association for Cancer Research. CELLULAR RESISTANCE Cellular Fractionation. [195mPt]CDDP was received with a specific activity of 145-165 mCi/mmol. The half-life of "5mPt is 4.02 days, and TO CDDP niques. The cells were washed three times with PBS and suspended in 50 HIMsodium phosphate buffer, pH 6.5. The cell suspensions were kept at 4"( ', disrupted by sonication (Sonifier 200; Bronson, Inc.), and therefore experiments were carried out immediately upon receipt of the drug. Two million SCC-25 and SCC-25/CP cells/plate were incubated centrifuged at 16,000 x g for 30 min. GST activity in the supernatant with 100 /iM [l95mPt]CDDP in media without sera for 60 min at 37°C was measured by the method of Habig et al. (42) using 1 mM 1-chloro2,4-dinitrobenzene as the electrophilic substrate. GST activity is ex in humidified air with 8% CO2. Cells were washed six times with PBS, pressed as nanomoles of GSH-l-chloro-2,4-dinitrobenzene conjugate fresh medium containing serum was added, and the plates were replaced in a 37°C/8%CO2 incubator for 0, 3, 6, or 24 h. At the end of each formed per min per mg protein. The results presented are from cells harvested on three separate occasions. time period, cells were suspended using 0.5% EDTA/0.125% trypsin, centrifugea at 500 x g, resuspended in water, and lysed by sonication Cell extracts were analyzed by isoelectric focusing on 0.5 mm 5% polyacrylamide gels through a pH 3-10 ampholine gradient (Serva on ice. Biochemicals). After focusing was complete, the gels were divided The fractionation procedure of Sharma and Edwards (34) was per formed at 4°Cand the final pellets were resuspended in water. Lysed according to pH into 0.75-cm wide segments from which GST activity cells were centrifuged at 1000 x g for 10 min and the pellet resuspended was eluted and measured as described above (42). The isoelectric points in 0.25 M sucrose/1.8 HIMCaCl2/l% Triton X-100 solution. An equal of the GST isoenzymes were determined by use of marker proteins (Serva Biochemicals) and pH measurement of adjacent gel segments. volume of 0.34 M sucrose/0.18 imi CaCl2 solution was added to the bottom of the tube, pushing up the lighter solution. After cent rifugat ion The major isozyme was defined as containing more than 70% of the total GST activity recovered from the gel. at 600 x g for 10 min, the nuclear pellet was obtained. The supernatant Alkaline Elation. Alkaline elution was performed by standard pro from the first 1000 x g spin was centrifuged at 3500 x g for 10 min to cedures (5, 6, 43-45). SCC-25 or SCC-25/CP cells (2.75 x IO5)were obtain the mitochondria! pellet. The second supernatant was centri labeled with 1.25 nCi of [l4C]thymidine (New England Nuclear, Boston, fuged at 16,000 x g for 20 min to obtain the h soso mal pellet. The MA) in Dulbecco-Vogt modified Eagle's minimum essential medium third supernatant was centrifuged at 100,000 x g for 60 min to obtain supplemented with 5% FBS for approximately three doublings, after the microsomal pellet. This final supernatant contained the cytosol. which the [uC]thymidine was removed and the cells washed. Drugs The purity of the subcellular fractions from each cell line was checked by electron microscopy. 195mPtcontent was determined using a Beckman were added to the cells in fresh medium without serum and then incubated for 60 min at 37°C.The cells were washed with PBS, fresh Gamma 4000 counter. The amounts of ["5mPt]CDDP were determined medium plus 5% FBS was added, and incubation at 37°Cwas resumed. by comparison to standards. Each point is the mean of duplicate At various time intervals cells were removed with 0.1% EDTA. Onedeterminations in two independent experiments. The range of duplicate half of each group of cells was irradiated on ice with 600 rads using a determinations was ±3%(SE) and the range of experimental values Gammacell 40 (Atomic Energy of Canada, Ltd.). Approximately 5 x was ±8%. IO5cells/group were placed onto an alkaline elution filter (2.0 nm pore Protein Determinations. Protein was measured using a modified size) (Millipore, Bedford, MA). As an internal control, the same number biuret assay (Sigma Diagnostics, Sigma Chemical Co., St. Louis, MO) of'l'H |thy midine-labeled LI 210 cells irradiated with 150 rads were also following the cellular fractionation procedure described above. placed onto each filter. Cells were first washed with cold PBS, then Sulfhydryl Measurements. SCC-25 and SCC-25/CP cells in expo nential growth (4 x 10') were lysed in 2 ml of 5% perchloric acid. The lysed with 3 ml of 0.2% sodium dodecylsarkosine/2 M NaCl/0.04 M EDTA, pH 10, which was allowed to flow through by gravity. To study protein was removed with 5 M potassium phosphate by centrifugation DNA-DNA interactions, 0.5 mg/ml proteinase K (Sigma) was added at 1000 x g for 3 min at 5°C.The supernatant was neutralized then to the lysis solution, and incubation on filters was allowed to proceed passed through a 0.2-itm filter before derivatization. Glutathione was for 60 min at room temperature. Alkaline elution was carried out using assayed at four dilutions of the supernatants. 2% tetrapropylammonium hydroxide (Fisher Scientifïc)/0.025 M For the fluorescence assay, 0.1 ml of the sample was added to 3 ml EDTA at a rate of 2.4 ml/h in the dark. Fractions were collected at 90of 0. l M potassium phosphate buffer containing 5 HIMEDTA (pH 8), min intervals and were assayed for radioactivity after adding 12 ml of then 0.15 ml of an OPT (Aldrich Chemical Co., Milwaukee, WI) Aquasol (Dupont/New England Nuclear). The remaining DNA on the solution (1 mg/ml OPT in methanol) was added. The derivatization filters was removed by treatment with 0.4 ml of l N HC1 for 60 min at was allowed to continue for 15 min at room temperature in the dark. 65°C,and the solution was neutralized with 2.5 ml of 0.4 N NaOH Fluorescence was measured with an excitation wavelength of 350 nm before Aquasol addition. Samples were counted on an LS 7000 Beckand an emission wavelength of 420 nm. The calibration curve was linear man scintillation counter. Each point was measured in three independ from 0.05-50 nmol of GSH/ml (35, 36). The data shown are the mean ent experiments. Cross-linking factor was calculated as of five determinations. Glutathione was also determined by the method of Fahey et al. (37). /irradiated L°g(—....control\ 1 Glutathione was derivatized by monobromobimane and quantitated by control V peak height on high-performance liquid chromatography. Non-protein and total sulfhydryl contents were determined using a modification of /irradiated drug \ the Ellman method (38-41). SCC-25 and SCC-25/CP cells (IO7) were L0gl control j lysed in 0.02 M EDTA (6 ml). For non-protein sulfhydryl determina tion, the cell lysate (5 ml) was treated with 10% trichloroacetic acid (5 ml), then centrifuged at 500 x g for 5 min at 5°C.The supernatant (4 RESULTS ml) was adjusted to pH 8.9 with 0.2 M Tris buffer. Ellman's reagent [0.01 M 5,5'-dithiobis(2-nitrobenzoic acid)] in methanol (Aldrich) (12.5 The SCC-25/CP cell line was developed by repeated treat Ml/ml sample) was added and color was allowed to develop for 30 min at room temperature. Total sulfhydryl content was determined by adding cell lysate (0.5 ml) to 0.2 M Tris buffer ( 1.5 ml, pH 8.2) followed by 0.1 ml of 0.01 M 5,5'-dithiobis(2-nitrobenzoic acid) in methanol. ment with escalating concentrations of CDDP and is generally not cross-resistant to alkylating agents of the nitrogen mustard and nitrosourea classes as determined by median inhibitory concentration ratios (30). However, it was not known whether After bringing the sample volume to 3 ml with methanol, the color was the SCC-25/CP line would be resistant to other platinum allowed to develop for 30 min at room temperature. All of the samples complexes. The survival of SCC-25 and SCC-25/CP cells ex were filtered (0.45 nm) and absorbance was read at 412 nm. Protein posed to various concentrations of CDDP and two secondsulfhydryl content was determined from the difference between the total generation platinum complexes, carboplatin and ipropiatin, is sulfhydryl content and non-protein sulfhydryl content. The measure shown in Fig. 1. A 30-fold higher concentration of CDDP is ment was repeated three times. required to kill 90% of SCC-25/CP cells compared to SCC-25 Glutathione Transferase Measurements. SCC-25 and SCC-25/CP cells were maintained in cell culture and harvested by standard tech cells. Carboplatin and ipropiatin are less potent cytotoxic agents 389 Downloaded from cancerres.aacrjournals.org on April 28, 2017. © 1987 American Association for Cancer Research. CELLULAR RESISTANCE TO CDDP 100 300 500 1000 100 300 500 1000 100 300 500 DRUG CONCENTRATION, 1000 yU Fig. 1. Survival of SCC-25 (•)and SCC-25/CP (O) cells treated with various doses of CDDP (A), carboplatin (/(), or iproplatin (('). Points, means of three independent determinations ±SE (bars). 30.0 0.03 036 24 0 3 6 Time (hrs) Fig. 2. Subcellular distribution of "5mPt in SCC-25 and SCC-25/CP cells. Cells were exposed to 100 ^M ("5"Pt]CDDP for 1 h. The zero time is immediately after drug removal. A, cytosol; •,nuclei; O, mitochondria; •,lysosomes; D, microsomes. SCC-25/CP cells as indicated by the levels of platinum in these cells after 1 h exposure to the drug provides these cells with one mechanism of resistance. However, the amount of platinum which is found in the nuclei of SCC-25/CP cells is lower than expected on the basis of the plasma membrane barrier alone. Therefore, we examined the cytosol of both cell lines for pos sible differences which could account for the increased differ ential in nuclear platinum content. The non-protein sulfhydryl content of both cell lines was assayed using OPT derivatization and fluorescence measurement for total non-protein sulfhydryls and monobromobimane derivatization, high-performance liq uid chromatography, and fluorescence detection for GSH (Ta ble 1) (35-37). There was no difference in the non-protein sulfhydryl content between the cell lines, and the ratio of GSH levels between the cell lines was 1.11 ±0.13. However, the protein sulfhydryl content, as measured by Ellman's procedure, indicated that the SCC-25/CP cell line has approximately a 2fold increase in protein sulfhydryl content compared to the SCC-25 cell line (38-41). Metallothioncins, which contain about 30% cysteine, can comprise a large percentage of cellular protein sulfhydryl content. Resistance to the cytotoxic effects of cadmium chloride has been used as an indicator for an increased metallothionein content (46-49). The survival of SCC-25 and SCC-25/CP cells exposed to various concentra tions of cadmium chloride for 1 h is shown in Fig. 3. The SCC25/CP cell line is about 2-fold resistant to cadmium chloride at 50% cell kill and about 2.5-fold resistant at 1 log kill compared to the SCC-25 cell line. This finding correlates very well with the increase in protein sulfhydryl content of the SCC25/CP cells. In addition to metallothionein binding, conjugation of CDDP to GSH by glutathione-S-transferase could account for the reduced nuclear content of CDDP. GST activity in crude cytoplasmic extracts of both cell lines was measured and GST activity was found to be 2- to 3-fold higher in the SCC-25/CP cells. The isoelectric point of the principal GST isozyme was 4.8 in both the SCC-25 and SCC-25/CP cell lines. This suggests induction of the predominant isozyme present in the parent cell line rather than induction of a new or previously undetectable isozyme. It is believed that the major mechanism of the cytotoxicity of CDDP is the formation of cross-links in DNA (2, 5, 6). The formation of DNA cross-links by CDDP in the SCC-25 and SCC-25/CP cells was assessed using alkaline elution (45) at three concentrations (50, 10, and 2 /IM)after 1 h exposure to the drug (Fig. 4). The formation of DNA cross-links was followed for 48 h. Overall, there was greater cross-link forma tion in the SCC-25 than in the SCC-25/CP cell line. Although the formation of cross-links in the SCC-25/CP cell line appears to plateau after 24 h, there was no evidence of removal of the cross-links formed up to 48 h. When the levels of cross-links formed at the various drug concentrations are compared, the cross-linking factor in SCC-25/CP cells is approximately 2fold lower than that of the SCC-25 cell line at the same drug concentration. than CDDP. The SCC-25/CP cell line is approximately 10fold resistant to carboplatin and 9-fold resistant to iproplatin. We conclude, therefore, that SCC-25/CP cells are cross-resist ant to these platinum complexes which are structurally related to CDDP. Using [l95mPt]CDDP,we examined the levels of platinum in whole cells and cellular fractions of both the SCC-25 and SCC25/CP cells after l h exposure to 100 /¿M drug (Fig. 2) (34). Measurements of platinum levels in the various cellular frac tions were made both immediately after removal of drug and at various times over the following 24 h. The SCC-25 cells took up 30 pmol of platinum/IO6 cells in 1 h; 64% of the drug was in the nucleus and 21% in the cytosol. Each of the other cellular fractions (lysosomes, microsomes, and mitochondria) contained 5% of the total platinum taken up. The SCC-25/CP cells took up 7 pmol of platinum/10* cells; of this, 41% was in the nucleus and 33% in the cytosol. Each of the other cellular fractions from this cell line contained 8-9% of the platinum taken up. The SCC-25/CP cells (0.391 mg protein/106 cells) are some what smaller than the SCC-25 cells (0.457 mg/106 cells); how ever the nuclei in both cell lines contain 0.058 mg protein/IO6 cells. Therefore, the SCC-25 cell nuclei contained 331 pmol of DISCUSSION platinum/mg protein and the cytosol 21 pmol of platinum/mg protein, whereas the SCC-25/CP cell nuclei contained 47 pmol CDDP has gained wide clinical use as an antineoplastic of platinum/mg protein and the cytosol 8.1 pmol/mg protein. alkylating agent (1, 50). The goal of this study is to understand The release of drug from both cell lines followed a similar the mechanism(s) by which the SCC-25/CP human squamous course and was most rapid over the first 6 h. After 24 h, the carcinoma cell line is resistant to CDDP. These mechanisms SCC-25 nuclei retained 26 pmol of platinum/mg protein. The may also account for the cross-resistance to carboplatin and iproplatin in this cell line. Cross-resistance with CDDP ana SCC-25/CP nuclei retained 5.5 pmol of platinum/mg protein. logues has previously been reported in a human ovarian cancer Reduced uptake of CDDP through the plasma membrane of 390 Downloaded from cancerres.aacrjournals.org on April 28, 2017. © 1987 American Association for Cancer Research. CELLULAR RESISTANCE TO CDDP Table 1 Protein and non-protein sulfliydryl content and glutathione transferase specific activity in SCC-25 and SCC-25/CP cells nmol/106 cells Glutathione transferase activity* Non-protein Protein Isoelectric sulfhydryl" sulfhydryl* point** (nmol/min) Cell line 25±2<(55±5/ 231 ± 25 (503 ± 55)* SCC-25 23 ±3 (48 ±7) 4.8 SCC-25/CP 447 ±73(1145 ±186) 21 ±3 (54 ±7) 39 ±2 (100 ±5) 4.8 • Measured by fluorescence emission at 420 nm of an OPT derivative. 0 Measured by the difference between total sulfhydryl content and non-protein sulfhydryl content using Ellman's method. Absorbance was measured at 412 nm. '' Activity determined by absorption at 340 nm of product formed by the reaction of GSH with l-chloro-2,4-dinhroben/ene. d For the principal glutathione transferase isozyme present in these cells. ' Mean ±S.E. ^Numbers in parentheses, nmol per 10* cells per mg protein. * Numbers in parentheses, nmol per mg protein. 1.0 CdCI2 0.1 0.01 0.001 50100 500 1000 Drug Concentration uMolar Fig. 3. Survival of SCC-25 (•)and SCC-25/CP (O) cells treated with various doses of cadmium chloride. Points, means of three independent determinations ± SE (bars). 14 SCC-25 ,-12 o o «10 O) -E 8 JÃC m S 4j 0 , t- C 3 12 24 48 Time, Hrs. Fig. 4. DNA cross-linking in SCC-25 and SCC-25/CP cells at various times after drug exposure. The concentrations of CDDP used were: •,50; O, 10; and •.2 /IM. The cross-linking factor was calculated according to the method of Kohn et al. (43). Data were derived from three independent experiments ±SE (bars). cell line made resistant to CDDP (51). The cellular distribution of platinum following treatment with CDDP has been described for cells in culture (52, 53) and in several tissues in vivo (34, 54, 55). Using analytical electron microscopy, Khan and Sadler (52) found that CDDP was predominantly localized in the nucleolus and the inner side of the nuclear membrane. In nucleosomes, using a fluorescent probe, histone H3 as well as DNA were found to be targets for CDDP (53). The intracellular localization of platinum in liver, kidney, and other tissues has been measured by atomic absorption (34, 55) and scanning transmission electron microscopy in conjunction with X-ray probe microanalysis (54). In these tissues, high percentages of platinum were found in the cytosol bound to metallothioneinlike proteins and in lysosomes in the kidney tubules. Studies of L1210 cells resistant to CDDP have provided evidence that the drug is transported into the cells by an amino acid transport system (11-13). Plasma membrane alteration is a common mechanism of cellular resistance to al kviating agents (11-13, 15-18) and to a wide variety of other drugs and toxins (56-58). We have found that both SCC-25 and SCC-25/CP cells con centrate platinum in the nuclei to some degree; however, there is a difference in platinum levels in the parent line compared to the CDDP resistant line, implying an alteration in the plasma membrane of the SCC-25/CP cells leading to decreased plati num levels in that cell line under the same exposure conditions as the parent line. There also appears to be cytosolic changes in the SCC-25/ CP cell line. Although the levels of the non-protein sulfhydryl, GSH, are the same in both cell lines, there is an increase in protein sulfhydryl content and an increase in GST in the SCC25/CP cell line. When the SCC-25 and SCC-25/CP cell lines were treated with BSO, the levels of GSH in both cell lines decreased similarly. The response of both cell lines to the cytotoxic actions of CDDP remained unaffected by the BSO treatment.4 Similar results with BSO have been reported re cently in two human ovarian carcinoma cell lines 2.5- to 3-fold resistant to CDDP (59). Increased protein sulfhydryl content and cadmium chloride resistance indicate that the SCC-25/CP cell line may have increased levels of metallothionein-like pro teins. These proteins have been induced acutely by exposure to cadmium, zinc, and glucocorticoid hormones through increased transcription (60,61) or chronically through gene amplification (46). Although CDDP has not been shown to induce metallothionein (47, 48), cells which have a high content of metallothionein are resistant to CDDP (49), and CDDP has been shown to bind to metallothioneins both in vitro and in vivo (62, 63). In conclusion, the mechanism of resistance of the SCC-25/ CP cell line to CDDP is multifactorial: (a) there are reduced intracellular levels of platinum in the SCC-25/CP compared to SCC-25 cells, implying altered plasma membrane properties in the resistant line; (b) there is a 2-fold increase in protein sulfhydryl content, a 2-fold increase in GST, and a 2-fold reduction in sensitivity to CdCl2, implying altered cytosolic binding and metabolism of CDDP in the resistant line; and (c) there is a reduced level of DNA cross-linking by CDDP in the SCC-25/CP line compared to the parent line. In addition to these changes which we have measured, there may be other alterations in the SCC-25/CP cell line which result in the 30fold resistance observed. ' E. Frei III, B. A. Teicher, and C. A. Cucchi, upublished observations. 391 Downloaded from cancerres.aacrjournals.org on April 28, 2017. © 1987 American Association for Cancer Research. CELLULAR RESISTANCE TO CDDP REFERENCES 1. Prestayko,A. 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Characterization of a Human Squamous Carcinoma Cell Line Resistant to cis-Diamminedichloroplatinum(II) Beverly A. Teicher, Sylvia A. Holden, Michael J. Kelley, et al. Cancer Res 1987;47:388-393. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/47/2/388 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on April 28, 2017. © 1987 American Association for Cancer Research.