Increased sensitivity to 1-β-d-arabinofuranosylcytosine in P388 murine leukemic cells resistant to etoposide

Leukemia Research Vol, 13, No. 1, pp. 39~2, 1989. Printed in Great Britain.

0145-2126/89$3.00 + .00 PergamonPressplc

I N C R E A S E D SENSITIVITY TO 1-[3-DA R A B I N O F U R A N O S Y L C Y T O S I N E IN P388 M U R I N E L E U K E M I C CELLS RESISTANT TO ETOPOSIDE* MASAMUNE H I G A S H I G A W A , t MASARU I D o , t T O S H I K 1 0 H K U B O , t HAJIME K A W A S A K I , t HITOSHI K A M I Y A , t MINORU S A K U R A I , t KIYOSU TANIGUCHI~ a n d MINORU HAMAZAKI:~

t Department of Pediatrics, Mie University School of Medicine, 2-174 Edobashi, Tsu-city, Mie-ken, 514, Japan and * Department of Clinical Pathology, Shizuoka Children's Hospital, 860 Urushiyama, Shizuoka-city, Shizuoka-ken, 420, Japan

(Received 2 May 1988. Revision accepted 20 August 1988) Ahstraet--A variant P388 murine leukemic cell resistant to 4'-demethylepipodophyllotoxin-9-(4,6-Oethylidine)-13-D-glucopyranoside (etoposide) (VP-16-213) was cloned. The variant P388/VP-16 cell line was 159-fold resistant to VP-16. We found that this variant P388/VP-16 cell line showed collateral drug sensitivity to 1-13-D-arabinofuranosylcytosine(Ara-C), determined by comparing the 50% inhibitory concentrations in 48-h growth inhibition assay. To clarify the mechanism of this increased sensitivity to Ara-C, we quantified the deoxyribonucleoside triphosphate pools and 1-13-Darabinofuranosylcytosine triphosphate(Ara-CTP) using high-performance liquid chromatography in the parent and drug-resistant sublines of P388 ceils. The analysis of deoxyribonucleoside triphosphate pools revealed that the pyrimidine triphosphate pools were significantly decreased in the P388/VP16 cell line and the Ara-CTP concentration of two variant cell lines were not significantly different. The Ara-CTP/dCTP ratio was significantly increased in P388/VP-16 cells. These data suggest that the inhibition of the dCTP de-novo pathway and the preservation of the dCTP salvage pathway in P388/VP-16 cells might correlate with the increased sensitivity to Ara-C.

Key words: Collateral drug sensitivity, multiple drug resistant, Ara-C, deoxyribonucleotides pools, Ara-C'TP, etoposide, P388 murine leukemic cell.

INTRODUCTION

lymphomas [1]. The development of resistance of tumor cells to anticancer drugs is one of the major problems related to effective cancer chemotherapy. The simultaneous resistance to several classes of drugs in vivo and in vitro after exposure to a chemically dissimilar agent is termed multiple drug resistance [2-4]. The most effective strategy is the choice of non-cross-resistant drugs when the tumors develop the resistance to the treated drugs. We report here that the increased sensitivity to Ara-C followed by the decrease of the deoxyribonucleoside triphosphate pools and the increase of the A r a - C T P / d C T P ratio in P388 murine leukemic cells resistant to etoposide.

VP-16-213 IS A semi-synthetic derivative of podophyllotoxin. The clinical efficacy of this compound has been shown against a spectrum of human tumors, such as small cell lung cancer, testicular carcinoma, acute myelogenous leukemia and

* Supported in part by a Grant-in-Aid (6040050) for scientific research from the Ministry of Education, Science and Culture of Japan. Abbreviations: VP-16, 4'-demethylepipodophyllotoxin9- (4,6 - O- ethylidine) - 13-D - glucopyranoside (etoposide); Ara-C, 1-13-D-arabinofuranosylcytosine; Ara-CTP, 1-[3-Darabinofuranosylcytosine 5'-triphosphate; dCTP, deoxycytidine 5'-triphosphate; dTTP, deoxythymidine 5'-triphosphate; dA TP, deoxyadenosine 5'-triphosphate; dGTP, deoxyguanosine 5'-triphosphate; FCS, fetal calf serum; RPMI 1640, Roswell Park Memorial Institute Medium 1640; TCA, trichloroacetic acid; P-388/VP-16, P388 murine leukemic cells resistant to etoposide; IC5o, concentration of drug required for 50% inhibition of cell growth. Correspondence to: Dr Masamune Higashigawa, Department of Pediatrics, Mie University School of Medicine, 2174 Edobashi, Tsu-city, Mieken, 514, Japan.

MATERIALS AND METHODS

Chemicals 1,1,2-trichlorotrifluoroethane(freon) was purchased from E. Merk AG., Darmstadt, Federal Republic of Germany and tri-n-octylamine(alamine) was purchased from Tokyo Chemical Industry Co. Ltd, Tokyo, Japan. Disodium hydrogen phosphate (Na2HPO4) was purchased from Nakarai Chemicals Co. Ltd, Kyoto, Japan. 39

40

M. HIGASHIGAWAet al.

Tumor cell lines P388/VP-16 cells were developed by treatment of BDF1 mice bearing the P388 ascites cells with 8 mg VP-16-213/ kg body weight on days 1 and 5 after i.p. implantation of 106 P388 cells, as described by Wilkoff and Dulmadge [5]. P388 parent cells and P388/VP-16 cells were transferred to in-vitro culture and maintained in RPMI 1640 medium supplemented with 10% FCS and 201xM 2-mercaptoethanol. 3.0 × 107 cells in logarithmic growth phase were used in each experiment.

Flowcytometric cell cycle analysis Cell cycle distribution was calculated using flow cytometry after DNA staining in a propidium iodine solution. The cells were run in a EPICS-C flow cytometer (Coulter Electronics Inc., Hialeah, FL) equipped with a 2-W argon laser operating at 200 mW at 488 nm in light regulation mode. 2 × 104 cells of both cell lines were examined. The DNA histogram was analysed by the sum of broadened rectangle method.

Quantitation of drug effects The growth-inhibitory effects of drugs were assessed by plating cells in multiwell dishes (Falcon 3047) at a final density of 1-2 x 105/ml. The ICs0 was defined as the concentration of drug that inhibits the 48-h cell growth by 50%, compared with untreated controls. The extent of increase in ICs0 was determined by dividing the ICs0 for the resistant cells by that for the controls.

Sample preparation for HPLC The deoxyribonucleoside triphosphate pools were extracted by a modified method of Khym and Santi [6, 7]. The P388 parent and P388/VP-16 cells were incubated with Ara-C at the concentration of 10 ~tM at 37°C for 3 h under 5% CO2, 95% air. The cells were washed with ice cold 0.9% saline. The pellet of 3 × 107 cells was added with 200 ~tl of 0.6 N TCA in Eppendorf's micro test tube, and was vortexed and kept at 0°C for 10min. After centrifugation (15,000 g, for i min at 4°C), the supernatant was separated and added with 300 I~1of cold freon-alamine (1,1,2-trichlorotrifluoroethane:tri-n-octylamine = 7.81: 2.19) and was vortexed for 5 min. After centrifugation, 100 ~tl of the aqueous upper portion was separated and, following the addition of 20 ~tl of 0.2M NaIO4, was incubated at 37°C for 2 min. Then 2 ~tl of 1 M rhamnose and

30 M1of methylamine were added to the reaction mixture and was incubated at 37°C for 30 min. The samples were stored at -20°C until applied to the HPLC.

Determination of deoxyribonucleoside triphosphate pools and Ara-CTP. The separation of the deoxyribonucleoside triphosphate pools and Ara-CTP was achieved using a Waters' A L G / GPG 204 HPLC equipped with two 6000 A pumps, a 440 UV detector, a 660 solvent programmer, a 710 B sample processor and a 730 data module. An isocratic separation was performed using a TSK gel D E A L 2SW column (Tosoh Ltd, Japan) with 0.06 M disodium hydrogen phosphate with 20% acetonitrile at pH 6.6. The flow rate was 0.7 ml/min. The quantitation was done by means of peak height measurement. RESULTS

Characterization of P388/Parent and P388/VP-16 cell lines W h e n growing in R P M I 1640 m e d i u m supp l e m e n t e d with 10% FCS and 20 ~tM of 2-merc a p t o e t h a n o l , the P 3 8 8 / P a r e n t and P388/VP-16 cell lines had doubling times of 9.9 and 12.6 h, respectively (Table 1). Figure 1 shows the D N A distribution patterns in P 3 8 8 / P a r e n t and P388/VP-16 cell lines. T h e flow cytometric D N A - a n a l y s i s revealed that the percentages o f the S-phase of P 3 8 8 / P a r e n t and P388/ VP-16 cell lines were 54.0 and 39.5%, respectively (Fig. 1). T h e deoxyribonucleoside triphosphate pools in P 3 8 8 / P a r e n t and P 3 8 8 / V P - 1 6 cells are shown in Table 2. T h e d C T P and d A T P pools of P388/VP-16

TABLE 1. DOUBLING OF TIME TWO VARIANTS OF P388 CELLS

Variant

Doubling time (h)

P388/Parent P388/VP-16

9.9 12.6

TABLE 2. DEOXYRIBONUCLEOSIDETRIPHOSPHATELEVELSIN P388/PARENT AND P388/VP16 CELLS Concentration of deoxyribonucleoside triphosphate pools (pmol/1 × 106 cells)

P388/Parent P388/VP-16

d-TTP*

d-CTPt

d-ATP$

d-GTP*

57.2 +- 1.2 50.6 +- 0.0

38.2 -+ 0.0 25.2 -+ 0.0

26.7 +- 1.1 15.8 +- 0.0

5.3 -+ 1.7 3.5 +- 0.0

The acid soluble fractions were extracted with 0.6N TCA, and analysed by highperformance liquid chromatography as described in "Materials and Methods". Values are the mean -- S.D. of two independent experiments. * Not significantly different (Student's t-test). t p dCMP 4 :CTP4

Id. . . . . ~ ~XdT T P:

.~dTDP~

ara C dCyd

UTP~ pathway I

t. dTMP,~

kinase

way and the preservation of the dCTP salvage pathway in P388/VP-16 cells might correlate with the increased sensitivity to Ara-C.

~U D P + - - ~ UM P J' dUOP dU~MP

FIG. 2. The de-nooo pathway of the pyrimidine triphosphates and the formation of Ara-CTP by the salvage pathway. A decrease in dCTP concentration due to inhibition of its de-novo synthesis results in enhancement of the salvage pathway.

collateral drug sensitivity to Ara-C, determined by comparing the 50% inhibitory concentrations in 48h growth inhibition assay. One of the mechanisms of action of Ara-C is thought that Ara-C undergoes phosphorylation to form Ara-CTP, which competitively inhibits D N A polymerase in opposition to the normal substrate dCTP [10]. Therefore to clarify the mechanism of this increased sensitivity, we quantified the deoxynucleosides triphosphates pools and the level of Ara-CTP in the parent cells and the P388/VP-16 cells. The analysis of deoxynucleosides triphosphates pools revealed that the pyrimidine triphosphates pools were significantly decreased but the A r a - C T P / d C T P ratio were significantly increased in P388/VP-16 cells. Plagemann et al. [11] reported that the intracellular dCTP concentration was normally high enough to cause severe feedback inhibition of deoxycytidine kinase. But a decrease in dCTP concentration due to inhibition of its de-novo synthesis resulted in enhancement of the salvage pathway (Fig. 2). The affinity constant for Ara-C is higher than the physiologic substrate deoxycytidine [12]. However, the deoxycytidine kinase is strongly inhibited by dCTP, but weakly inhibited by Ara-CTP, thus allowing accumulation of the Ara-C nucleotide to higher concentration [13]. Therefore we think that the AraC T P / d C T P ratio correlates with the cytotoxicity of Ara-C. The causes of depression of dATP in P388/ VP-16 cells have been unclear whether the secondary changes are due to the depression of pyrimidine pools or not. Although the P388/VP-16 cell line had a longer doubling time and a smaller S-phase than P388/Parent cell line, the concentration of Ara-CTP of P388/VP-16 cells was not significantly different from that of the P388/Parent cells. Therefore the salvage pathway of dCTP was preserved in P388/VP16 cells. These results support our tentative conclusion that the inhibition of the dCTP de-novo path-

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