Properties of Amino Acid Transport Systems in ... - Cancer Research

[CANCER RESEARCH 46, 3445-3448, July 1986]

Properties of Amino Acid Transport Systems in K562 Cells Sensitive and Resistant tocw-Diamminedichloroplatinum(II)1 S. Shionoya,2 Y. Lu, and K. J. Scanlon3 Section of Biochemical Pharmacology, Department of Medical Oncology, City of Hope National Medical Center, Duarte, California 91010

ABSTRACT When K562 cells were made resistant to cisplatin their neutral amino acid transport systems changed. K562 cells cloned in cisplatin developed a 6.7-fold resistance to the drug. The generation times for K562 cells sensitive (S) and resistant (DDP) to cisplatin were similar. The initial uptake and rapid efflux of cisplatin were similar in both cell lines. However, they differed in their sodium dependent neutral amino acid transport properties. In K562S cells the sodium-dependent uptake for methylaminoisobutyric acid was significantly inhibited by threonine (K¡ = 10.3 HIM),but in K562DDP the uptake of neutral amino acids was significantly lower, and methylaminoisobutyric acid uptake was mini mally inhibited by threonine. When K562DDP cells were grown in the absence of cisplatin for 8 weeks, their amino acid transport properties reverted to those of K562S cells. In K562 cells, changes in plasma membrane essential amino acid transport systems were concomitant to both the development of resistance and the redevelopment of sensitivity to cisplatin. Therefore, human malignant cell sensitivity and resistance to cisplatin may be related to membrane nutrient transport systems.

INTRODUCTION Cisplatin [m-diamminedichloroplatinum(II)] has been a po tent anticancer chemotherapeutic agent, both as a single agent and in combination regimens (1). Its antitumor activities may be explained in part by the cross-linking reactions involving DNA and protein (2-4) and its effects with cell surface nucleic acids (5) or the plasma membrane (6). Cisplatin has been shown previously to inhibit the uptake of neutral amino acids into the LI210 cells via the ASC-Iike transport system, while in the LI 210 resistant cell line cisplatin was a poor inhibitor of amino acid uptake (6, 7). This permanent alteration of ASC-like transport system in LI210 cells was also associated with a stable resistance to cisplatin (7). In contrast to these mouse cell studies, there are few reports of human cell lines with a stable resistance to only cisplatin (8). Thus, the definitive mechanisms of resistance of human cells to cisplatin remain to be solved. Although the resistance of K562 cells to cisplatin was not stable, data presented in this study suggest that changes in the neutral amino acid transport systems might be correlated to cisplatin cytotoxicity. These interactions with the plasma membrane might account for part of the antitumor activities of cisplatin in human tumors.

the Drug Synthesis and Chemistry Branch, National Cancer Institute, Bethesda, MD. Cell Culture. K562 cells (K562S) (9) were obtained from Dr. Berlino at Yale University and maintained in Fischer's medium (Grand Island Biological Co., Grand Island, NY) supplemented with penicillin (62.9 mg/1), streptomycin (100 mg/ml), and 10% swine serum (Hyclone, Ogden, UT) (10). Cisplatin-resistant cells (K562DDP) were obtained by cloning in soft agar with 16.5 JIMcisplatin and 15% swine serum. The K562DDP cells were maintained either by continuous exposure to 3.3 /IM cisplatin in suspension culture or in the absence of drug. Cell growth studies were determined by soft agar cloning of K562 cells in the continuous presence of cisplatin, and colonies were scored at day 12-14(7). Uptake and Efflux of Radioactive Cisplatin. The uptake and efflux of ["""Pt]cisplatin into K562S and K562DDP cells were determined with 0.6-1.0 x io" cells/ml in Earle's balanced salt solution as described previously (6). The cell pellet was dissolved in 6.5 ml of Scinti-Verse (Fisher Scientific, Fair Lawn, NJ) and counted by Liquid Scintillation System LS3801 (Beckman Instruments, Inc., Somerset, NJ). Amino Acid Transport Studies. The net sodium-dependent amino acid transport was determined by subtracting the sodium-independent transport from the total transport in the presence of sodium (6). Incubation medium for determining sodium-independent transport was Earle's balanced salt solution in which 116 mM choline chloride was substituted for 116 HIMNaCl. Uptake of labeled amino acids (threonine and MeAIB) was determined by measuring the incorporation of radio activity into the cell pellet. Log-phase cells were resuspended in the incubation medium to a density of 2.5 x 10' cells/ml, and 200-Ml aliquots were withdrawn at timed intervals after adding labeled amino acids (6). The cell pellets were separated immediately by centrifugation through a gradient of an oil (2 parts silicone: 1 part dinonylphthalate) (11) into 12% perchloric acid, dissolved in 6.5 ml of Scinti-Verse, and counted by Liquid Scintillation System LS3801. The initial velocity was determined by linear-least squares fit of the uptake versus time. The kinetic parameters (Kt and Vra„) were determined by an iterative non-linear least-squares program to the direct Michaelis-Menten equa tion. The data were not weighed (12). K, has been defined as the concentration of the amino acid substrate required for one-half satura tion of a mediated-transport system (13).

RESULTS

Tumor Cell Growth in the Presence of Cisplatin. Cisplatin inhibited the colony formation of K562S and K562DDP cells with an ED5o (concentration which induces 50% inhibition of MATERIALS AND METHODS cell proliferation) of 0.67 and 4.5 MM,respectively (Fig. 1). The Chemicals. L-[C-3H]Threonine(20 Ci/mmol) were purchased from K562DDP cells were 6.7-fold resistant to cisplatin, and the Amersham, Arlington Heights, IL. MeAIB4 (51.6 mCi/mmol) was resistance was unstable. Within 4 weeks of being grown in drug purchased from New England Nuclear, Boston, MA. ["5mPt]Cisplatin free medium, the K562DDP cells were again equally as sensitive (145 mCi/mmol) was obtained from Oak Ridge National Laboratories, to cisplatin as were the parent cells (Fig. 2). The generation Oak Ridge, TN (6). Cisplatin (NSF no. 119875-1)was obtained from time for K562S and K562DDP cells in culture was 22.2 ±0.8 (SD) and 25.4 ±2.3 h, respectively. Received 9/26/85; revised 2/3/86, 4/1/86; accepted 4/2/86. Initial Uptake and Efflux of [l95mPt]Cisplatin. The uptake of

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 was supported by the American Cancer Society (Grant 256) and the Chemotherapy Foundation, Inc. 2 Present address: Saitama Medical Center, Kawagoe-shi, Saitama 350, Japan. 3 Scholar of the Leukemia Society of America. To whom requests for reprints should be addressed. 4 The abbreviations used is: MeAIB, a-[l-14C]niethylaminoisobutyric acid.

cisplatin (1 and 5 MM)during the first 7.5 min was similar in both K562S and K562DDP cells (Fig. 3). The uptake of cispla tin (25 MM)into K562S and K562DDP cells increased with time over 75 min (Figs. 4 and 5). In the resistant cells, the initial binding of cisplatin to the cell was 13% less when compared to the sensitive cells (Figs. 4 and 5). The net uptake

3445

MEMBRANE

PROPERTIES

OF K562 CELLS

O

10

5

l/Thr (mW1)

Fig. 7. Threonine uptake in K562S and K562DDP cells. Sodium dependent uptake of threonine at various concentrations (100-1200 UM) was measured in K562S (O) and K562DDP (•)cells. V was expressed as pmol/103 cells/0.5 min. Bars, SE.

3.0 K* -I033IÕ

051-nM

2.0 I/V

0

10

20

30 40 50 TIME(min)

60

70

Fig. 5. The uptake and the efflux of cisplatin from K562DDP cells. The K562DDP (•)cells were incubated at 37'C or 24'C separately with 25 IM cisplatin. The uptake was measured over 75 min. The efflux ( ) of cisplatin (•)was measured at 20 and 40 min by diluting the K562 cells 20-fold with drug free medium at either 37'C or 24*C. Bars, SE.

-50 -40 -30 -20 -IO

O

IO 20

30 40

50

Thr (mM) Fig. 8. Mi-AIH uptake in the presence of unlabeled threonine in K562S cells. Sodium dependent uptake of labeled MeAlB (0.2, 0.4. 0.6, and 0.8 mM) was measured in the presence of unlabeled threonine (0, 10, 25, and 50 mM).

-dependent-DDP/È/"s• 2016I/V128/*No

Drug free medto: OK562S •K562DDP a K562 DOP 4 weeks OK562DDP 8 weeks

100

^-/\/-^-8-404

/

161/MeAIB

8

12

(mW1) Fig. 6. Initial MeAlB uptake in K562S and K562DDP cells. Sodium depend ent initial uptake (1 min) of MeAlB at various concentrations (75 -600 pM) was measured in K562S (O) and K562DDP (•)cells. V was expressed as pmol/103 cells/0.5 min. Bars, SE.

cisplatin (10 ^M) for 15 min at 37°Cthere was a decrease in the sodium dependent uptake of threonine, MeAlB, and methionine by 57.6, 33.1, and 28.3%, respectively (Table 1). Neu tral amino acid uptake in K562DDP cells was not significantly influenced by cisplatin (Table 1). DISCUSSION In this study, cisplatin cytotoxicity in K562 cells did not correlate with drug permeability but with the expression of the essential sodium dependent amino acid transport systems.

10

25

UNLABELED Thr (mM)

Fig. 9. MeAlB competition by threonine in K.562S and K562DDP cells. Sodium dependent uptake of 500 tiM labeled MeAlB was measured for 4 min in the presence of 0, 10,25, or 50 mM unlabeled threonine in K562S or K562DDP cells. The control (100%) was the uptake of MeAlB alone in the K562S cells. Bars, SE.

K562DDP cells were established by soft agar cloning in the presence of cisplatin. However, these cells could revert to the drug sensitive parental phenotype within 4-8 weeks if they were maintained in the drug-free medium. This finding contrasts with L1210 murine leukemia cells, which had a stable resistance

3447

MEMBRANE

PROPERTIES

Table I Influence ofcisplatin on amino acid uptake in K562 cells KS62S and K.562DDP cells were preincubated for IS min with cisplatin (10 ...M) at 37'C. The initial uptake for methionine in KS62S and KS62DDP was 2.8 and 1.2 pmol/103 cells/min, respectively. The initial uptake for MAIB and threonine was obtained from Figs. 6 and 7, respectively. The sodium dependent uptake was measured as described previously (6, 7). inhibition of sodium de uptakeK562S56.7 pendent

Amino acid MM)ThreonineMeAIBMethionineCisplatin101010% (500 14*33.1 ± 828.3±

±10K562DDP