THE JOURNAL OF BIOLOGICAL CHEMISTHY Vol. 261, No. 17, Issue of June 15, pp. 7762-7770,1986 Printed in U S A .

Multiple Drug-resistant Human KB Carcinoma Cells Independently Selected for High-level Resistance to Colchicine, Adriamycin, or Vinblastine Show Changes in Expression of Specific Proteins*

(Received for publication, June 21, 1985)

Ding-wu Shen, Carol Cardarelli, Jaulang HwangS, Marilyn Cornwell, Nancy Richert, Shunsuke Ishii, Ira Pastan, and Michael M. GottesmanQ From the Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892

We have established four cell lines derived from the human KB carcinoma cell line which express high- level multiple drug resistance. One of these lines was selected for resistance to colchicine, one was selected €or resistance to colchicine in the presence of the tumor promoter, mezerein, one for resistance to vinblastine, and one for resistance to adriamycin. All of these cell lines are cross-resistant to the other selective agents. The development of multidrug resistance in these cul- tured human carcinoma cells is associated with a lim- ited number of specific protein alterations revealed by high resolution two-dimensional gel electrophoresis and Western blot analysis. These protein alterations in multidrug-resistant lines include the decreased preva- lence of members of a family of proteins of molecular mass 70,000 to 80,000 daltons, PI 4.8-5.0, the in- creased synthesis of a protein of molecular mass 21,000 daltons, PI 5.0, in the colchicine-resistant cell lines only, and the increased expression of a 170,000- dalton protein in membrane preparations from all of the resistant cells. The loss of the 70,000- to 80,000- dalton proteins in the multidrug-resistant lines, which can also be demonstrated by immunoprecipitation of these proteins with specific antisera, is associated with a loss of translatable mRNA for these proteins. These studies suggest that only a limited number of protein changes occur in multidrug-resistant cell lines.

The development of simultaneous resistance to multiple drugs appears to be a major impediment to the successful chemotherapy of human tumors. A variety of tissue culture systems have been established over the past several years to study the biochemical, physiologic, and genetic bases of alter- ations that result in the development of multidrug resistance. These systems include multidrug-resistant hamster cells and mouse cells (1-4) and human leukemic and sarcoma cells (5, 6). We have recently demonstrated the development of mul- tiple drug resistance in a human KB epidermoid carcinoma cell line selected for resistance to colchicine ( 7 ) .

Many of these multidrug-resistant cell lines have exhibited alterations in the expression of high molecular weight glyco- proteins on their cell surfaces such as the increased expression of a 170,000-dalton P-glycoprotein in multidrug-resistant

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

3 Recipient of an Anna Fuller Fellowship. 0 To whom correspondence should be addressed: NCI, NIH, Bldg.

37, Rm. 2E18, Bethesda, MD 20892.

Chinese hamster ovary (CHO)’ cells (8-12) increased gp150,OOO on Chinese hamster lung cells (13) and increased gp180,OOO on human leukemic cells (14). The presence of the P-glycoprotein has been correlated with reduced accumulation of colchicine in the CHO mutants (15). We have also observed decreased accumulation of drugs in our multidrug-resistant KB cell lines (16). Other protein changes in multidrug-resist- ant cell lines have also been recently described, including the increased synthesis of a protein of 19,000 daltons in a variety of cell types selected for resistance to high levels of vincristine (17) and decreased synthesis of two proteins of molecular mass 72,000 and 75,000 in colchicine-resistant KB cells (18).

We undertook the current studies in order to establish independent multidrug-resistant cell lines from the same KB parent used for our initial studies (7) . We have used these cell lines to catalog the changes in specific protein synthesis associated with the development of multidrug resistance. We describe here the selection and isolation of KB cells that are resistant to high levels of either colchicine, vinblastine, or adriamycin. All these lines show multidrug resistance. An analysis of protein synthesis in these cell lines by high reso- lution two-dimensional gel electrophoresis and Western blot analysis shows only a limited number of specific protein alterations, some of which are shared by all cell lines and some of which are limited to a subset of the multidrug- resistant lines.

MATERIALS AND METHODS

Cell Lines and Cell Culture-KB-3-i, the parent cell line for the drug-resistant mutants described in this study, was derived from a single clone of human KB epidermoid carcinoma cells after two subclonings (7). The adriamycin-, vinblastine-, and colchicine-resist- ant sublines were selected with increasing concentrations of their respective drugs. In addition to colchicine, KB-ChR-8-5-MC3.5 (KB- MC3.5) and KB-MC3 were maintained in a fixed concentration of mezerein (0.05 pg/ml), a second stage tumor promoter which we used to promote gene amplification in a manner analogous to the use of 12-0-tetradecanoyl phorbol 13-acetate (19). KB-C1-Rl is a cloned spontaneous revertant of KB-ChR-8-5-11-24 (KB-C1) which was iso- lated from a population of KB-C1 cells grown in the absence of colchicine for two months.

The cell lines were all grown as monolayer cultures at 37 “C in 5% COz using Dulbecco’s modified Eagle’s medium with 4.5 g of glucose/ liter (Quality Biological) with 10% fetal calf serum (Whittaker, MA Bioproducts, Lot 4C005), L-glutamine (Whittaker), penicillin (50 units/ml) (Whittaker), and streptomycin (50 pgiml) (Whittaker). The drug-resistant sublines were maintained in their respective drug selection media up to the day of assay. The colchicine revertant was cultured in drug-free medium.

The abbreviations used are: CHO, Chinese hamster ovary; SDS- PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; IEF, isoelectric focusing; NEPHGE, nonequilibrium pH gradient electrophoretic analysis; EMS, ethyl methanesulfonate.

7762

Protein Expression in Multidrug-resistant Human Cells 7763

Drugs and Chemicals-Colchicine, adriamycin, vinblastine, and ethyl methanesulfonate (EMS) were Sigma products. The mezerein was from LC Services Corp. The drugs were dissolved in dimethyl sulfoxide (Aldrich) a t a concentration of 1 mg/ml.

Cell Suruiual by Colony Formation-The dose response curves of KB-3-1, its drug-resistant sublines, and its colchicine revertant were determined by plating 300 cells in 60-mm dishes in the absence of any drug. The various drugs from dimethyl sulfoxide stock solutions were added 16 h later. After incubation for 8-10 days at 37 "C, the colonies were stained with 0.5% methylene blue in 50% ethanol and counted. The Dlo value is the concentration of drug which reduced the cloning efficiency of the sublines to 10% of the control without drugs. Relative resistance was determined by dividing the Dl0 value of the resistant sublines and the revertant by the Dl0 value of the parental cells. The average cloning efficiency of KB-3-1 was 47%.

Isolation of Colchicine-, Adriamycin-, and Vinblastine-resistant Sublines-We have previously described the selection in four inde- pendent steps of KB-3-1 cells resistant to 1 pg/ml of colchicine (7). In the current work, we isolated KB cells resistant to higher levels of colchicine by stepwise increase of colchicine in the medium to 1.5, 2, 2.5, 3, 3.5, and 4 pg/ml.

We also isolated high-level colchicine-resistant lines from the low- level colchicine-resistant mutant KB-ChR-8-5 (7) in the presence of 0.05 pg/ml mezerein, with the intent of amplifying the genes respon- sible for the defect in this cell line. The colchicine concentration of the selection medium was gradually increased from 10 ng/ml to 3.5 pg/ml in mass cultured cell populations.

The first step adriamycin- and vinblastine-resistant sublines were isolated from KB-3-1 cells (1 X 106/100-mm dish) in 10 ml of medium using 250 pg/ml of EMS to enhance the mutation rate. After incu- bation for 24 h at 37 "C, the medium was removed, and the cells were incubated in EMS-free medium for five days to allow expression of the resistant phenotype. Adriamycin and vinblastine were then added to the medium in the concentrations shown in Fig. 1. Prior to assaying for drug resistance, the colonies were isolated with steel rings and maintained in their respective drug selection medium until they reached a population of lo6 cells.

The five colonies that were isolated at 10 ng/ml of adriamycin consisted of slow-growing, loosely arranged cells. Two of these colo- nies were tested for their resistance to adriamycin, vinblastine, and colchicine, and their cloning efficiencies were determined. KB-ADRR- 5A, the most adriamycin-resistant of the two lines, was chosen for subsequent stepwise increases of adriamycin concentrations in mass populations ranging from 20 ng/ml to 1.8 pg/ml.

A total of 10 vinblastine-resistant colonies were isolated from the dishes containing 1.5 ng/ml of drug in the selection medium. Two of these colonies were tested for their resistance to vinblastine, adria- mycin, and colchicine. The most drug-resistant colony, KB-VBLR-5, was chosen for a second selection at 3 ng/ml, and subsequently mass populations of this subclone (KB-VBLR-5-2) were grown in increasing concentrations of vinblastine up to 2 pg/ml.

KB-C1-R1 is a spontaneous revertant of KB-ChR-8-5-11-24 (3) that has been isolated and subcloned twice in the absence of colchi- cine. It has a relative resistance four times that of KB-3-1, the parental line, to colchicine.

Two-dimensional Gel Electrophoresis of ~5SlMethwnine-labeld Proteins-Cells were plated at a density of 3 X lo6 cells/100-mm dish in fresh Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. The following day, the cells were labeled in 5 ml of methionine-free modified Eagle's medium containing [35S]methionine (200 pCi/ml). When cells were labeled with [35S]methionine for 16 h, medium contained 5% fetal bovine serum. The cells were collected by centrifugation at 1200 rpm for 5 min and lysed in isoelectric focusing (IEF) lysis buffer (9.2 M urea, 2% Nonidet P-40, and a mixture of 0.8% pH 5-8 and 0.2% pH 3.5-10 Ampholines from LKB). Two- dimensional gel electrophoresis was carried out according to the method of O'Farrell (20). Immunoprecipitates to be analyzed by two- dimensional electrophoresis were prepared as previously described (18) and the Staphylococcus aureus pellets were eluted with IEF lysis buffer. Nonequilibrium pH gradient electrophoretic analysis (NEPHGE) was run according to the method of O'Farrell et al. (21).

Preparation of Poly(A)+ RNA-Total cellular RNA was isolated by guanidium thiocyanate extraction (22) and centrifugation through a cesium chloride cushion (23). Poly(A)+ RNA was prepared by oligo(dT)-cellulose chromatography (24).

Cell-free Translation and Immunoprecipitation-Rabbit reticulo- cyte lysate was used as recommended by the supplier (New England Nuclear). Poly(A)+ RNA (0.1 fig) was added for each 25-p1 translation

mixture. After translation for 60 min at 37"C, 3-pl aliquots were added to 25 pl of PAGE-sample buffer (0.07 M Tris-HC1, pH 6.8, 11.2% glycerol, 3% SDS, 0.01% Promphenol blue, 5% P-mercaptoeth- anol) and immediately boiled at 100 "C for 2 min. For the immuno- precipitation, the remaining 22-pl reaction mixture was added to 1 ml of buffer A (0.5% Nonidet P-40, 150 mM NaC1, 20 mM Tris-HC1, pH 7.2, 0.018% SDS, 0.5% Trasylol). Normal rabbit serum (10 was added, and after 1 h at 4 "C, 50 pl of heat-killed s. aureus were added and left for a further 30 min. The bacteria were pelleted in an Eppendorf microfuge. Finally, 3 pl of affinity-purified antibody (about 20 pg of pure antibody) was added to the clear supernatant, left overnight at 4 "C, and then precipitated by adding 50 pl of S. aureus for 30 min. The bacteria were washed twice in 1 ml of buffer A, once in 1 ml of buffer A containing 1 M NaC1, and once more in 1 ml of buffer A and boiled for 2 min in PAGE-sample buffer. The total protein products and immunoprecipitates were analyzed by SDS- PAGE and fluorography.

Western Blot Analysis for a 170,000-Dalton Glycoprotein Associated with Multidrug Resistance-The expression of the 170,000-dalton glycoprotein (~170) shown to be associated with multidrug resistance in CHO cells (12) was determined by Western blotting according to the method of Kartner et al. (25). The parental CHO cell line AUX- B1 and the multidrug-resistant line C5 were kindly provided by Dr. Victor Ling (University of Toronto) and grown as previously de- scribed (12). Membrane pellets were prepared by centrifuging cell extracts a t 100,000 X g as described (12). The human KB carcinoma cell lines KB-3-1 and KB-C4 were used to make membrane vesicles as described by Lever (26) and were used as the source of membrane proteins for these cell lines. Membrane proteins were fractionated on 5% polyacrylamide gels as described by Fairbanks et al. (27) and transferred to nitrocellulose paper as previously described (28). p170 was detected using a lZ5I-labeled monoclonal antibody, C219, gener- ously provided by Dr. Ling (25).

RESULTS

Isolation and Cross-resistance of Multidrug-resistant Cell Lines-We have previously described the selection of colchi- cine-resistant KB cells in four steps (7). In this paper, we describe the further selection of these colchicine-resistant lines (Fig. 1). We also placed the second step mutants, KB- ChR-8-5, resistant only to 10 ng/ml colchicine, in the presence of nontoxic concentrations of mezerein (0.05 pg/ml) and increasing amounts of colchicine to select cells highly resist- ant to colchicine as shown in Fig. 1. To obtain adriamycin- or vinblastine-resistant lines, it was necessary to mutagenize the cells to get colonies in the first selection step, but subse- quent steps yielded either colonies or mass populations able to grow in increasing concentrations of drug without diffi- culty. Clonal populations are designated with an R superscript and a clone number (i.e. KB-VBLR-5), whereas uncloned populations are given letter designations (i.e. KB-C4 grows in 4 pg/ml colchicine).

Fig. 2 shows the cloning efficiency of the four cell popula- tions, the parent cell line (KB-3-l), and a colchicine revertant in increasing concentrations of the three drugs. Each of the drug-resistant cell lines showed cross-resistance to each of the other drugs. Relative resistances of each cell line at several steps in the selection process are tabulated in Table I. In general, the lines are most resistant to the drugs against which they were selected. However, at the highest levels of selection, the vinblastine-resistant population (KB-V1) is more resistant to adriamycin than vinblastine. In general, no matter what the selecting agent, relatively higher vinblastine resistance appears before adriamycin resistance or colchicine resistance.

The revertant cell line was obtained by allowing KB-C1 cells to grow in the absence of colchicine. Drug-sensitive subclones were obtained and recloned in the absence of col- chicine to yield the cloned cell line KB-C1-Rl. As can be seen from Fig. 2 and Table I, this revertant line has lost most of its resistance but is still resistant to all three drugs to a small

7764 Protein Expression in Multidrug-resistant Human Cells Colchicine Resistant Adriimycin Resistant Vinblastine Resistant

I FIG. 1. Flow diagram showing the

derivation of the multidrug-resist- ant HB cell lines isolated for this study. The parent cell line was a KB subline designated KB-3-1. ChR, colchi- cine-resistant subclones; ADRR, adria- mycin-resistant subclones; VBL', vin- blastine-resistant subclones; C, colchi- cine-resistant populations; MC, colchi- cine-resistant populations selected in the

I K B - A D R R - ~ ~ +

t + +

KB-A.@

KB-A.05

KB-A.l

KB-A.5 I

EMS, lOng/rnl adr EMS, 5ng/ml col

EMS. 10ng/rnl COI 1 KE-Ch -8

KB-Chfl.8-5 + mezemin. / \ . 0 5 & n l

KB-ChRS571 KB-MC.15

KB-ChR-%511-24 KE-MC.3

iKB;Clt KB-MC.5

t t + +

I

6 steps, .CGpg/ml inCrwnemS

.%steps, .1 @/ml inwemenk

presence of 0.05 ig/ml mezerein; A, adri- t amycin-resistant populations; V, vin- blastine-resistant populations (in each case where a letter alone is used, the number following the letter refers to the KE-U.5 selecting concentrations of drug in p g l 1 ml); EMS, mutagenesis by ethyl meth- K B - U anesulfonate. t

+

KB-Cl.5

KE-C2 + t

KBX3.5

KE-C4

KB-MC.7 t

KB"C1 t t t t t

KE-MC1.5

KB-MC2.5

KB-MC3

KE-MC3.5

KE-A.7 t

KB-A.8

KB-A1

KB-A1.6

4

+ t

Ke"V1

TABLE I Relative resistance of multidrug-resistant KB cell lines

Cell line" Relative resistanceb

Colchicine Vinblastine Adriamycin

KB-3-1 1 1 1 Colchicine-resistant

KB-ChR-8-5 3.8 6.3 3.2 KB-C1 263 96 162 KB-C1.5 324 142 ND KB-C2.5 487 206 141 KB-C4 1750 159 254 KB-MC3.5 62 108 135

Colchicine-revertant

Vinblastine-resistant KB-C1-R1 6 4 3

KB-VBLR-5 1.8 2.8 1.8 KB-VBLR-5-2 2.6 23 5.1 KB-V.5 80 425 169 KB-V1 171 213 422

Adriamycin-resistant KB-ADRR-5A 1 1.9 2 KB-A.05 9.4 38 31 KB-A1 19 43 97

Relative resistance is expressed as the Dlo of the resistant line divided by the Dl,, of KB-3-1 (parental, drug-sensitive) cells.

"he numbers following the initials, C (colchicine), V (vinblas- tine), or A (adriamycin), are the selecting concentrations of drugs in pg/ml for uncloned populations. Cloned cells are designated by Ch (colchicine), VBL (vinblastine), or ADR (adriamycin) followed by an isolate number.

COLCHICINE (ng/ml)

VINBLASTINE hg/rnll

extent (similar to KB-ChR-8-5). Two-dimensional Gel Electrophoresis of Methionine-labeled

Proteins in Colchicine-resistant Cells-To examine the protein alterations in colchicine-resistant human KB carcinoma cell lines, cell proteins were radiolabeled with [35S]methionine for 16 h and analyzed by high resolution two-dimensional gel electrophoresis (Figs. 3-5). The colchicine-resistant cells have decreased amounts of [35S]methionine-labeled proteins in the molecular mass range of 70,000-80,000 Da, with PI values of 4.8-5.0 when compared with the parental and revertant lines (Fig. 3, panels A-D). This result is consistent with our pre- vious observation that colchicine-resistant KB cells have de- creased amounts of 75,000- and 72,000-Da glycoproteins (18). The decreased amount of these proteins seen on two-dimen- sional gels correlates with the increase in colchicine resistance of the KB cells. In addition to the protein changes in the 70,000-80,000-Da region, colchicine-resistant cells were also found to have increased amounts of protein with a molecular

Protein Expression in Multidrug-resistant Human Cells 7765

Neutral IEF Acid PI - 6;O 5:s 5,O 4:5 Mr.

I

FIG. 3. Two-dimensional equilib- rium gel analysis of [36S]methio- nine-labeled protein from parental, colchicine-resistant and revertant cell lines. Cells were labeled with [35S] methionine for 16 h. Radiolabeled ah- quots (1 X lo6 cpm, 30-50 pg of protein) were subjected to isoelectric focusing with a pH gradient from 7 to 4 in the first dimension then run in the second dimension on a 5-15% linear gradient acrylamide gel. Completed gels were ex- posed to x-ray film after fluorography at -70 "C for 16 h. A, KB-3-1 (parental); B, KB-C1-R1 (revertant); C, KB-C1; D, KB-C4.

-200kd

-92kd

-68kd

-45kd

-31kd

Neutral I EF Acid

FIG. 4. Two-dimensional equilibrium gel analysis of [36S]methionine-labeled protein from parental, adriamycin-, and vinblastine-resistant cell lines. Cells were labeled with [35S]methionine for 16 h. Electro- phoretic analysis was as described in the legend to Fig. 3. A, KB-3-1; B, KB-A1.7; C, KB-V1.

weight of 21,000, PI = 5.0, designated C21. The increase in C21 seen in colchicine-resistant cells was proportional to their levels of colchicine resistance. The amount of C21 in revertant cells (Fig. 3, panel B ) was similar to that found in the parental cells. Adriamycin- and vinblastine-resistant cell lines also showed decreased amounts of 70,000-80,000-dalton proteins on two-dimensional gels (Fig. 4) and by immunoprecipitation with antiserum (data not shown).

To confirm that the 70,000-80,000-Da proteins on the two- dimensional gels are identical to those previously reported (18), immunoprecipitations were performed as previously de- scribed (18) and analyzed on two-dimensional gels. The im- munoprecipitate showed that the precipitated proteins comi- grate with the proteins shown with arrows in Figs. 3 and 4 (data not shown).

Further experiments were done to examine basic protein

7766

Mr.

92kd- -

68kd-

45kd -

31kd-

C

Protein Expression in Multidrug-resistant Human Cells

9.5 9.0 8.5 8.0 7.5 I I I I I

FIG. 5. Nonequilibrium pH gradient electrophoretic analysis (NEPHGE) of [35S]methionine labeled proteins from parental, colchicine-resistant, and revertant cell lines. Cells were labeled with [35S] methionine for 16 h. Aliquots (1 X lo6 cpm, 30-50 pg of protein) of [35S]methionine-labeled cell extracts were subjected to NEPHGE with a pH gradient from 7 to 10.5 in the first dimension. Each first dimension gel was then run in the second dimension on a 5-15% linear gradient acrylamide gel. Completed gels were exposed to x-ray film after fluorography at -70 "C for 16 h. A , KB-3-1; B, KB-C1-R1; C , KB-C1; D, KB-C4.

v,

CI, 0 I

GI rn

v

changes as seen by nonequilibrium pH gradient electropho- resis (NEPHGE) followed by SDS-PAGE (Fig. 5 ) . A single protein change was found at molecular mass 45,000 Da, PI = 8.0; a decrease in this spot was seen in the colchicine-selected cell lines, KB-C1 and KB-C4 (Fig. 5, C and D).

Two-dimensional Gel Electrophoresis of p5S]Methionine Pulse-labeled Proteins from Drug-resistant KB cells-To de- termine whether the protein changes described above were found in other drug-resistant cell lines, we analyzed [35S] methionine-labeled proteins in independently isolated colchi- cine-, vinblastine-, and adriamycin-resistant cell populations, all of which were cross-resistant to a variety of drugs (Fig. 2). To determine whether the protein changes were due to altered stability or altered rate of synthesis, cells were pulse-labeled with [35S]methionine for 30 min. As in the 16-h labeling experiments, we observed an increased amount of C21 in KB- C4 and KB-MC3 but not in cells selected with adriamycin (KB-A1.7) or selected with vinblastine (KB-VI) (Fig. 6). This result suggests that the overproduction of C21 in KB-C4 and KB-MC3 is due to an increase in the rate of synthesis and that the increased synthesis of C21 is specific for KB cells selected for colchicine resistance.

The 70,000-80,000-Da proteins were not readily detected in extracts from parental or drug-resistant cell lines pulse- labeled for 30 min. Because these proteins are glycosylated (18), it is likely that they are synthesized as lower molecular weight precursors during the 30-min pulse period (see below) and are not visible in the 70,000-80,000-Da region of the gel.

Fig. 7 shows NEPHGE followed by SDS-PAGE of these pulse-labeled cell lines to examine the basic proteins. Several protein spots are seen to be altered in these gels. Spot 2 is the 45,000, PI 8.0 protein which is decreased in KB-C1 and KB- C4 (Fig. 3, C and D ) and it is seen to be decreased in the adriamycin-resistant line as well (Fig. 7E). Spot 1 ( M , 48,000, PI 7.5) is increased in adriamycin- and vinblastine-resistant lines (Fig. 7, E and F). Spots 3 and 4 (Mr 48,000, PI 8.4; and M , 40,000, PI 9, respectively) are increased only in the most resistant colchicine selected line, KB-C4 (Fig. 7C). Spot 5 (Mr 34,000, PI 7.4) was decreased only in KB-C4 (Fig. 7C). Spot 6 (Mr 32,000, PI 7.8) was increased in KB-C4, KB-MC3, and KB-V1 (Fig. 7, C, D, and F ) . Spots 8 and 9 ( M , 64,000, PI 8.6; and M , 32,000, PI 8.5, respectively) were increased only in KB-MC3. These changes were found to be reproducible, but no changes seen in these NEPHGE gels affected all the multidrug-resistant lines.

Translation and Immunoprecipitation of p72 and p75- Since we could not identify the 70,000-80,000 molecular weight proteins in our pulse-labeling experiments, we were interested in whether reduced amounts of these proteins were related to their decreased synthesis. To examine this question, we determined whether the amount of translatable mRNA species for these proteins were decreased in the drug-resistant mutant. A rabbit reticulocyte lysate system was used to syn- thesize these proteins using poly(A)+ RNA of parental KB-3- 1, the colchicine-resistant mutant, KB-C2.5, and the revertant KB-C1-R1. The fluorogram shows that the total translation

Protein Expression in Multidrug-resistant Human Cells 7767

Neutral I EF Acid c

I A B - C

c

FIG. 6. Two-dimensional equilib- rium gel analysis of [36S]methionine pulse-labeled proteins from inde- pendently isolated multidrug-resist- ant cell lines selected in colchicine, vinblastine, and adriamycin. Cells were labeled with [35S]methionine for 30 min. Electrophoretic analysis was as de- scribed in the legend to Fig. 3. A , KB-3- 1; B, KB-C1-R1; C, KB-C4; D, KB-MC3; E, KB-A1.7; F, KB-V1. . -

I

0 . -. .

- 0 " -

0 . i- - -

0

products programmed with poly(A)+ RNA of these three cell lines are very similar (Fig. 8A). The polyclonal antibody (prepared against KB cell membranes) which recognizes the 72 and 75-kDa proteins (18), as well as a 36-kDa protein, immunoprecipitated three major species synthesized in uitro, with molecular weights of 63, 60, and 36 kDa (Fig. 8B). Proteins of similar molecular weight were immunoprecipi- tated from extracts of tunicamycin-treated cells (data not shown). Therefore, the translation products of 63 and 60 kDa are most likely the unglycosylated precursor polypeptides of the 75- and 72-kDa proteins. There is a significant decrease in both the 63- and 60-kDa protein in translation products programmed with poly(A)+ RNA from KB-C2.5. The amount of 63- and 60-kDa proteins synthesized from the revertant KB-C1-R1 poly(A)+ RNA are higher than those from KB- C2.5 poly(A)+ RNA and almost the same as those from KB- 3-1 poly(A)+ RNA. These data show that the amount of translatable mRNA species for 75- and 72-kDa proteins are decreased in the drug-resistant mutant KB-C2.5 and, there- fore, loss of these proteins in the colchicine-resistant lines is likely to be due to decreased synthesis.

Detection of a 170,000-Dalton Protein in Multidrug-resist- ant KB Cells-Although alterations in the high molecular weight region of our two-dimensional gels were not observed, such changes might be missed because of the lack of solubility

of high molecular weight membrane glycoproteins. To test for the presence of the 170,000-dalton molecular weight glycopro- tein (p170) described by Riordan and Ling (12) in the mem- branes of multidrug-resistant CHO cells, we probed Western blots of membrane preparations with a monoclonal anti-pl70 antibody (25). As shown in Fig. 9, both multidrug-resistant CHO cells and KB-C4 cells express easily detectable amounts of p170, whereas parental CHO and KB cells had no detect- able p170. Similar amounts of p170 were found in membrane preparations of KB-A1 and KB-V1 multidrug-resistant mu- tants (data not shown).

DISCUSSION

The development of simultaneous resistance to multiple drugs when cells are exposed to a single selective agent ap- pears to be a common phenomenon among tissue culture cells (1, 4, 5, 7, 29) and may be an important mechanism for the development of resistance to chemotherapeutic agents in tu- mors. We have developed an in uitro model to determine the mechanism of multidrug resistance in human carcinoma cells (7). In this work, we describe the isolation of four lines of human KB carcinoma cells selected for high-level resistance to colchicine, adriamycin, or vinblastine. Each of the cell lines shows cross-resistance to each of the other drugs and to various unrelated drugs, such as actinomycin D (data not

7768 Protein Expression in Multidrug-resistant Human Cells

PI

FIG. 7. NEPHGE analysis of [36S] methionine pulse-labeled proteins from cell lines selected in colchicine, vinblastine, and adriamycin. Cells were labeled with [35S]methionine for 30 min. Electrophoretic analysis was as de- scribed in the legend to Fig. 4. A, KB-3- 1; B, KB-C1-R1; C, KB-C4; D, KB-MC3; E, KB-A1.7 F, KB-VI..

Mr. 9.5 9.0 8.5 8.0 7.5 92kd- 1 1 1 1 I

68kd-

45kd-

31kd-

Basic NEPHGE Neutral - ~~ -

I) -

v) 0 v)

3 R

c

shown). Although the absolute levels of cross-resistance vary somewhat with the selection conditions, a striking pattern of increasing cross-resistance is observed as cells are subjected to more stringent selective conditions.

High resolution two-dimensional gel analysis was used to screen our independently selected multidrug-resistant lines for altered proteins to determine which changes were unique to specific cell lines and which were common to all. One striking finding was that both qualitative and quantitative alterations in cell proteins were rare among these mutagenized cell populations which had been selected in many steps (see Fig. 1). Of 300-500 [35S]methionine-labeled protein spots which were easily resolved with the equilibrium and NEPHGE gel systems, only a few consistent changes were found. This result makes it unlikely that mutagenesis of these cell lines was producing multiple, independent changes in the cells which were responsible for the observed multiple drug resist- ance. The genetic data also strongly support the idea that the multidrug resistance phenotype is associated with a single locus or a small number of closely linked loci: 1) in all four selections, similar patterns of multidrug resistance developed coordinately; 2) the revertants we have analyzed (see Table I) have coordinately lost resistance to all tested agents; and 3) in somatic cell hybrids, resistance to each of the drugs is co-dominant, and in segregants of these hybrids, resistance to all drugs is coordinately lost (7).

One of the two prominent changes detected by our two- dimensional gel analysis is the loss of a family of proteins in the molecular weight range of 70,000-80,000 Da. The appear- ance of these spots on the two-dimensional gel of the parental KB cell extracts (Figs. 3 and 4) suggests that variable glyco-

sylation and perhaps sialylation could account for the increas- ing negative charge and increasing molecular weight. We have previously reported that a polyclonal antibody immunoprecip- itated two bands of molecular masses 72,000 and 75,000 Da from KB cells (18), which were decreased in the drug-resistant mutants. The series of spots observed in Figs. 3 and 4 corre- spond to these two proteins. The disappearance of these spots is seen in the colchicine-resistant lines, as previously reported (18), and also in the colchicine-resistant lines selected in the presence of mezerein and in the adriamycin- and vinblastine- resistant lines (Fig. 4). These proteins reappear in a revertant derived from the colchicine-resistant cells (Fig. 3B). These data strengthen the association of loss of this cell surface glycoprotein with the development of multidrug resistance in KB cell lines. Although these proteins are not observed in a short pulse-labeling experiment, this result is likely due to the high degree of modification of this protein which is probably time-dependent. We do, in fact, observe a loss of translatable mRNA for the 72,000 and 75,000 molecular weight proteins, indicating that their synthesis is probably reduced in the resistant cell lines. Whether the loss of this protein is responsible for the development of multidrug re- sistance in KB cells is not known, but it is clearly a useful marker for the development of this resistance.

A second major alteration in protein synthesis observed in this analysis is the increased expression of a protein of mo- lecular weight 21,000 and isoelectric point 5.0, termed C21. This increase in protein synthesis is seen only in the colchi- cine-resistant lines, KB-C1 and KB-C4, and in the colchicine- resistant line selected in the presence of mezerein (KB-MC3). One possible conclusion is that expression of this protein is

Protein Expression in Multidrug-resistant Human Cells 7769

A

94-

69-

W" 55" c 2 .. . ;.

a b c

B

+P17 0

- a b c

FIG. 8. Autoradiogram of [S6S]methionine-labeled proteins synthesized in a rabbit reticulocyte system. A , total protein products programmed with poly(A)+ RNA of KB-3-1 (lane a), KB- C2.5 (lane b), and KB-C1-Rl (lane c); B, immunoprecipitates of 60- and 63-kDa proteins synthesized by using poly(A)+ RNA of KB-3-1 (lane a), KB-C2.5 (lane b), and KB-C1-Rl (lane c). The number on the left shows the molecular mass (kilodalton) of marker proteins. All samples were run on 8% SDS-polyacrylamide gels.

related specifically to development of resistance to colchicine. It should be noted that the starting strain for selecting high- level resistance to colchicine in the presence of mezerein was KB-ChR-8-5, an early step in the independent selection for high-level resistance to colchicine in the absence of mezerein. Hence, it is possible that increased expression of C21, al- though not apparent in KB-ChR-8-5, may have been geneti- cally predetermined in this strain, and so its appearance in KB-MC3 might not be a truly independent event, although its increased expression certainly is. I t is noteworthy that Biedler and her co-workers (17) have previously described the increased expression of a 19,000-dalton protein with a PI of 5.7 in multidrug-resistant cells. We do not know if this is the same protein as C21 but have not observed an increase in the 19,000-dalton protein in our lines selected with vinblastine or adriamycin.

Several other protein changes were observed in some of our resistant lines compared to the parental or revertant cell lines on NEPHGE gels (Fig. 7). These changes frequently affected more than one of our multidrug-resistant lines, but no change affected all cell lines. There was a 2- to 3-fold decrease in an M , 45,000, PI 8.0, protein observed in all cell lines, with the possible exception of KB-VI, but the significance of this relatively small change is unknown.

Previous studies have reported a variety of biochemical alterations in multidrug-resistant cells, including the expres- sion on the cell surface of increased amounts of glycoproteins of molecular weights 150,000-180,000 (10, 11, 13, 14). Using ['4C]glucosamine labeling (7), iodination (data not shown), and precipitation with low affinity, polyclonal anti-pl70 anti- sera prepared from Chinese hamster p170 in our laboratory

FIG. 9. Expression of p170 in KB cells. Western blots were prepared according to the method of Kartner et al. (25) (see "Materials and Methods"). An '251-labeled monoclonal antibody ((2219) to p170 was used to identify p170 (indicated by arrow) in 100,000 X g mem- brane pellets from AUX-B1 cells (200 pg, lane I ) and C5 cells (200 pg, lane 2) or membrane vesicle preparations of KB-C4 cells (100 pg, lane 3) and KB-3-1 cells (100 pg, lane 4) .

(data not shown), we did not observe similar cell surface changes in our KB-C1 cells. However, using monoclonal an- tibodies generously provided by Dr. Victor Ling (25) and a Western blot procedure using a urea-containing polyacryl- amide gel system (27), we were able to detect p170 in our multidrug-resistant cells (Fig. 9):

The molecular basis for the protein alterations described in this work has not been established. Robertson et al. (30) and Meyers et al. (17) have described double minute chromosomes and homogeneously staining regions in drug-resistant cell lines, supporting a role for gene amplification in these resist- ant cells. Roninson and his co-workers (31) have recently described the amplification of a segment of DNA in two independently isolated multiply drug-resistant hamster cell lines. Using this hamster probe and a homologous human probe, we have been able to demonstrate amplification and expression of related sequences in all of the highly resistant cell lines described in this work (32, 33), and we also observe double minute chromosomes in many of our drug-resistant lines (34). In addition, using the ingel renaturation technique developed by Roninson (35) we have recently found that DNA sequences totalling at least 100 kilobases are amplified in all of our high-level multidrug-resistant cell lines (34). Whether

M. Cornwell, I. Pastan, and M. M. Gottesman, unpublished data.

7770 Protein Expression in Multl

the decreased expression of the 70,000-80,000-Da proteins is related to this gene amplification (as could occur if a gene repressor were amplified) or whether the increased expression of p170 and C21 is related to the amplified genes is uncertain at this time, but the cloning of the genes amplified in these cell lines should establish whether they encode the proteins described in this work.

Acknowledgments-We would like to thank Dr. Victor Ling for his generous gift of monoclonal antibody to p170, Joyce Sharrar for typing the manuscript, and Ray Steinberg for photographic assist- ance.

:drugresistant Human Cells C., Lazo, J. S., and Bertino, J. R., eds) pp. 453-482, Academic Press, New York

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17. Meyers, M. B., Spengler, B. A., Chang, T.-D., Melera, P. W., and Biedler, J. L. (1985) J. Cell Biol. 100 , 588-597

18. Richert, N., Akiyama, S.-i., Shen, D.-w., Gottesman, M. M., and Pastan, I. (1985) Proc. Natl. Acad. Sci. U. S. A. 82,2330-2333

19. Varshavsky, A. (1981) Cell 2 5 , 561-572 20. O’Farrell, P. H. (1975) J. Biol. Chem. 250,4007-4021 21. O’Farrell, P. Z., Goodman, H. M., and O’Farrell, P. H. (1977) Cell

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