[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-diam- minedichloroplatinum(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 10- fold 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 cyto- plasmic extracts was measured and found to be approximately 2- to 3- fold 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 antineo- plastic 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 (19- 22). 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 cyclophospha- mide-resistant human and rodent cell lines have increased levels of an aldehyde dehydrogenase that inactivates the aldophos- phamide 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(2- chloroethyl)-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-dihydroxo- bis(isopropylamine)platinum(IV) (iproplatin) were gifts from Johnson Matthey, Inc. (West Chester, PA). ["5mPt]cw-diamminedichloro- platinum(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 Research. on December 3, 2021. © 1987 American Association for Cancer cancerres.aacrjournals.org Downloaded from