Lack of a point mutation of human DNA topoisomerase II in multidrug-resistant anaplastic thyroid carcinoma cell lines

Cancer Letters 116 (1997) 33–39

Lack of a point mutation of human DNA topoisomerase II in multidrug-resistant anaplastic thyroid carcinoma cell lines Shoji Satake a, Isamu Sugawara b ,*, Masatoshi Watanabe b, Hiroshi Takami a b

a Department of Surgery, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi, Tokyo 171, Japan Department of Pathology, The Research Institute of Tuberculosis, 3-1-24 Matsuyama, Kiyose, Tokyo 204, Japan

Received 28 December 1996; revision received 6 February 1997; accepted 12 February 1997

Abstract DNA topoisomerases are major defined targets for a large variety of clinically important anticancer agents, including etoposide, adriamycin, and mitoxantrone. Mutations at amino acids 439, 450 and 803 of DNA topoisomerase II were examined in multiple anticancer drug-resistant anaplastic thyroid carcinomas (ten cell lines and three cancerous tissues) by reverse transcriptase-polymerase chain reaction (RT-PCR) and subsequent DNA sequencing. No mutation was found in these cell lines and tissues, but mdr1, mrp and/or lrp mRNA were expressed to a varying degree, and there was no significant difference in DNA topoisomerase IIa content among the cell lines and tissues as evaluated by Western blotting. Our experimental data indicate that overexpression of multidrug resistance-related mRNA is sufficient to confer drug resistance.  1997 Elsevier Science Ireland Ltd. Keywords: Multidrug resistance; DNA topoisomerase IIa; Anaplastic carcinoma; Thyroid

1. Introduction Although the incidence of anaplastic thyroid carcinoma is very low in Japan, less than 100 cases occurring annually, its prognosis is very poor, regardless of the results of various therapeutic trials [12,24]. We have reported a low frequency of mdr1 mRNA and P-glycoprotein expression and a high frequency of multi drug resistance-associated protein (MRP) expression in this cancer [18,19]. The 110-kDa lung resistance protein (LRP) is a vesicular protein involved in non-P-glycoprotein-mediated multidrug resistance [15,21]. DNA topoisomerases are major targets for a large variety of clinically important anticancer agents [2,5,14]. * Corresponding author. Fax: +81 424 924600.

Mammalian cells have two isozymes for type II topoisomerases, top2a and top2b [6,8]. All type II topoisomerases have regions of significant homology with each other [10]. Several mutations have been identified in human topoisomerase IIa from cell lines which are resistant to anti-topoisomerase II agents. So far, three mutations at amino acids 439, 450 and 803 of DNA topoisomerase IIa have been reported in anticancer agent-resistant cell lines [3,4,9,13]. This suggests that mutation of the equivalent amino acids in human topoisomerase IIa would produce a drug-resistant enzyme. These findings indicate that mutation of topoisomerase IIa as well as overexpression of mdr1 is closely associated with the mechanism of multidrug resistance. To address the issue of mutation confers a high level of drug resistance, anaplastic thyroid

0304-3835/97/$17.00  1997 Elsevier Science Ireland Ltd. All rights reserved P II S0304- 3835 (97 )0 4742- 3

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S. Satake et al. / Cancer Letters 116 (1997) 33–39

carcinoma cells were selected for study, since they are intrinsically resistant to various antineoplastic agents [20].

ity was reviewed by three immunopathologists, and similar results were obtained by each. 2.4. Reverse transcriptase-polymerase chain reaction (RT-PCR)

2. Materials and methods 2.1. Cell lines and tissues Ten anaplastic thyroid carcinoma cell lines (HTC/ C3, KKS-2, KTA-II, MC3, TC78, TCO-I, TTA-I, TTA-II, TTA-III and 8305C) and three specimens of anaplastic thyroid carcinoma tissues (50-year-old, male; 52-year-old, female; 70-year-old, female) were used in this study [20]. The tissues were snap-frozen and kept frozen until use. K562 was used as a control cell line for various assays because no normal thyroid cell line was available [18]. 2.2. Growth-inhibitory effects (IC50) of various anticancer agents on cell lines The IC50 was determined as described previously [16]. Briefly, cancer cells (3 × 105/ml) were cultured at 37°C for 5 h in 24-well culture plates containing 1.5 ml RPMI 1640 with 10% fetal calf serum. They were then treated with graded concentrations of adriamycin, bleomycin, cisplatin, etoposide or vincristine (0.001–10 mM). The cells were cultured in the presence of the drug and counted in a hemocytometer 3 days after drug treatment. Three wells were used for each drug concentration. The IC50 was determined by plotting the logarithm of the drug concentration versus the growth rate (expressed as a percentage of the control) of the treated cells. The initial cell number was subtracted in the calculation [23]. 2.3. Immunohistochemistry Immunohistochemistry was carried out as described elsewhere [11,16]. JSB-1 (MDR1), MRPm6 (MRP) and LRP-56 (LRP) were used as the first antibodies [21]. Immunoreactivity scoring was based on evaluation of cytospin and tissue preparations. Immunoreactivity was scored as negative when the cancer cells lacked immunostaining, and positive when they were immunostained. The immunoreactiv-

RNA was extracted from the specimens using a standard method. Five micrograms of purified cellular RNA was converted to single-stranded cDNA using a random primer, Moloney-MLV reverse transcriptase (Bethesda Research Laboratories, Gaithersburg, MD, USA) under reaction conditions described previously [1,20]. The cDNAs homologous with the mdr1, mrp, lrp and b2-microglobulin-positive strand RNA were subjected to 30 amplification cycles, using an automated thermal cycler [17]. The primers used in this study have been described previously [20,21]. The cycle conditions included a 65°C annealing step for 5 min, a 72°C extension step for 5 min, and a 94°C denaturation step for 1 min. A 308-bp DNA fragment for the mdr1, a 399-bp DNA fragment for the mrp, a 285-bp DNA fragment for the lrp and a 261-bp DNA fragment for the b2-microglobulin gene were detected under these conditions. A non-radioactive DNA sequencer (Pharmacia) was used to establish whether mutations at amino acids 439, 450 and 803 were present in the amplified 366-bp and 384-bp gene products of the DNA topoisomerase IIa gene (Table 1). Briefly, about 0.1 mmol/l FITC-labeled oligonucleotides were used for PCR under the same conditions as those described above. PCR was performed on total RNA. The sequence of FITC-labeled PCR products derived from total RNA was determined on-line on a nonradioactive DNA sequencer (Pharmacia) described previously. About 20% of each sequencing reaction product was run on a 7% polyacrylamide gel containing 8 mol/l urea. Products of the in vitro DNA amplification were detected on-line with the same device [1]. 2.5. Western blotting Western blotting was carried out as described previously [16]. Briefly, various an aplastic thyroid carcinoma cell lines and tissues were solubilized according to the method of Laemmli. The cells and tissues were solubilized with 500 ml of cell lysis buffer containing 1% Triton X-100, 1% sodium deoxycho-

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S. Satake et al. / Cancer Letters 116 (1997) 33–39 Table 1

Table 3

DNA topoisomerase IIa-specific primer sets used in this study

Immunohistochemical staining of cell lines with anti-MDR1, -MRP and -LRP monoclonal antibodies

Primer

Sequence (5′ to 3′)

Topo Topo Topo Topo

1251

II- 1 II-2 II-3 II-4

Expected amplification size

TAAGGCCCAAGTCCAGT1268T 384 bp 1617 CTGTCTAGTCCTGGTTC1634T 2206 CGGAATGACAAGCGAGAAG2225T 366 bp 2552 TACCACGACTTCCTTAGCC2571A

late, 0.1% sodium dodecylsulfate (SDS), 0.15 M NaCl, 50 mM Tris–HCl (pH 7.4), and 2 mM phenylmethylsulfonylfluoride (PMSF). After initial development of the solubilized proteins by SDS-PAGE, they were electrophoretically transferred onto a nitrocellulose membrane filter at a constant voltage of 50 V for 3 h. Thereafter, immunochemistry (ABC-PO method) was used for the detection of DNA topoisomerase IIa. An anti-DNA topoisomerase IIa polyclonal antibody (Biotrend Chemikallen GmbH, Koln, Germany) was used diluted 100-fold.

3. Results 3.1. In vitro cytotoxicity (IC50) As shown in Table 2, HTC/C3, TC-78, TCO1TTA-II, TTA-III and 8305C were significantly resistant to adriamycin, while HTC/C3, KKS-2, KTA-II,

Cell line

Immunoreactivitya of MDR1

HTC/3 KKS-2 KTA-II MC3 TC-78 TCO-1 TTA-I TTA-II TTA-III 8305C K562

MRP

+ + − − + + + − − + −

+ + + + + + + + + + −

LRP + + + − − + + + + + −

The immunostaining was classified as follows: −, negative immunostaining with anti-MDR1, -MRP and -LRP; +, positive immunostaining of cancer cells. a

MC3, TC-78, TTA-I and TTA-II were resistant to vincristine. HTC/C3 and TTA-II showed moderate resistance to bleomycin, but the other eight cell lines were not resistant. Five cell lines (HTC/C3, TTA-I, TTA-II, TTA-III and 8305C) showed resistance to cisplatin. All the cell lines were resistant to etoposide to varying degrees. 3.2. Immunocytochemistry As shown in Table 3 and Fig. 1, HTC/C3, KKS-2,

Table 2 Fifty percent growth-inhibitory concentration (IC50) of various anti-cancer agents against cell lines Cell line K562 HTC/C3 KKS-2 KTA -II MC3 TC-78 TCO-I TTA-I TTA-II TTA-III 8305C

Drugs (mM) Adriamycin

Bleomycin

Cisplatin

Etoposide

Vincristine

0.03 (1) 0.64 (213) 0.10 (3.3) 0.1 (3.3) 0.03 (1) 0.20 (6.66) 3.0 (100) 0.08 (2.66) 0.4 (13.3) 0.12 (6.66) 0.30 (10)

1.73 (1) 5.30 (3.1) 0.40 (0.2) 0.44 (0.25) 0.25 (0.14) 0.5 (0.29) 0.70 (0.40) 0.32 (0.18) 4.60 (2.65) 0.14 (0.08) 1.77 (1.02)

1.36 (1) 6.00 (4.4) 1.0 (0.73) 1.01 (0.74) 3.2 (2.35) 1.5 (1.10) 1.83 (1.34) 6.66 (4.9) 36.7 (27.0) 5.0 (3.7) 5.2 (14.4)

0.06 (1) 3.06 (51) 0.3 (5) 0.20 (3.3) 0.17 (2.83) 0.3 (5) 0.35 (5.83) 0.30 (5) 13.8 (230) 0.80 (13.3) 8.2 (136.6)

0.003 (1) 0.05 (16.7) 0.07 (20) 0.05 (16.7) 108 (36000) 0.08 (27) 0.01 (3.3) 56 (18667) 108 (36000) 0.01 (3.3) 0.006 (2)

Numbers in parentheses show relative resistance (cell line/K562).

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Fig. 1. Immunostaining of various cell lines with anti-MDR1 mAb (a,b), anti-MRP (c,d), anti-LRP (e,f). ABC-PO method. Counterstain with hematoxylin. × 79. (a) TCO-1. (b) MC3. (c) TCO-1. (d) MC3. (e) TCO-1. (f) MC3.

S. Satake et al. / Cancer Letters 116 (1997) 33–39

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TC-78, TCO-I, TTA-I, and 8305C were immunostained positively with JSB-1 monoclonal antibody (mAb). The other cell lines were immunostained negatively with mAb JSB-1. Ten cell lines except K562 used as a negative control were immunostained with mAb MRPm6. HTC/C3, KKS-2, KTA-II, TCO1, TTA-I, TTA-II, TTA-III and 8305C were immunostained intensely with mAb LRP-56. MC3 and TC78 were immunostained negatively with mAb LRP56. 3.3. RT-PCR data When mdr1 mRNA was evaluated by RT-PCR, HTC/C3, KKS-2, TC-78, TCO-1, TTA-I, and 8305C and cancer tissue 1 showed significant expression of mdr1 mRNA, but the level of expression was not as high as that of b2-microglobulin gene mRNA (Table 4). Mrp mRNA expression was observed in all of the anaplastic thyroid carcinoma cell lines and anaplastic thyroid carcinoma tissues. Lrp mRNA was expressed in all the cell lines except MC3, TC-78 and K562 and the cancer tissues (Fig. 2). DNA topoisomerase IIa mRNA was expressed uniformly and significantly in all of the cell lines and cancer tissues (Fig. 3). Table 4 mdr1, mrp, lrp and DNA topoisomerase IIa mRNA expression by RT-PCRa mRNA Cell line HTC/ C3 KKS-2 KTA-II MC3 TC-78 TCO-1 TTA-I TTA-II TTA-III 8305C K562 Cancer tissue 1 Cancer tissue 2 Cancer tissue 3

mdr1 + + − − + + + − − + − + − −

mrp

lrp

+ + + + + + + + + + − + + +

+ + + − − + + + + + − + + +

DNA topoisomerase IIa + + + + + + + + + + + + + +

−, none observed; +, strong (the amplified band visualized was less intense than that of half of the b2-microglobulin gene used as an internal control). a

Fig. 2. PCR products separated in 2% agarose gel. (a) M, size marker; lanes 1–7, mrp mRNA from HTC/C3, KKS-2, KTA-II, MC3, TC-78, TCO-1 and cancer tissue 1 (399 bp); lanes 8–14, mdr1 mRNA from HTC/C3, KKS-2. TC-78, TCO-1, TTA-I, 8305C and cancer tissue 1 (308 bp); lanes 15–17, b2 microglobulin mRNA from HTC/C3, KKS-2 and KTA-II (261 bp). (b) M, size marker; lanes 1–8, lrp mRNA from HTC/C3, KKS-2, KTA-II, TCO-1, TTA-I, TTA-II. TTA-III and cancer tissue 2 (285 bp); lanes 9–16, b2 microglobulin mRNA from the corresponding cell lines and tissue (261 bp).

3.4. DNA sequencing No mutations at amino acids 439, 450 and 803 were found in the amplified 366-bp and 384-bp DNA topoisomerase IIa gene products. Furthermore, no novel mutations were found in the gene products. 3.5. Western blotting The 170-kDa band was recognized in the ten anaplastic thyroid carcinoma cell lines and was slightly more intense than that in K562 (Fig. 4). The 180-kDa band corresponding to DNA topoisomerase IIb was hardly recognized in these cell lines.

Fig. 3. PCR products of DNA topoisomerase IIa separated in 2% agarose gel. M, size marker; lanes 1–13, DNA topoisomerase IIa mRNA from ten cell lines and three cancer tissues (384 bp).

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Fig. 4. Immunoblot of solubilized proteins from TTA-III, MC3, KKS-2, K562, KTA-II, TTA-I, cancer tissue 1 and 2. Immunoblotting was performed by ABC-PO method. The solubilized proteins (lanes 2, 4, 6, 8, 10, 12, 14 and 16) were immunostained with polyclonal anti-DNA topoisomerase IIa antibody, whereas the proteins (lanes 1, 3, 5, 7, 9, 11, 13 and 15) were immunostained with non-immune rabbit serum. The 170 kDa band is recognized.

4. Discussion Although our anaplastic thyroid carcinoma cell lines were resistant to various anti-cancer drugs to varying degrees, there were no mutations at amino acids 439, 450 and 803. Also, there was no significant difference in DNA topoisomerase IIa content between the anti-cancer drug-sensitive cell line K562 and the resistant anaplastic thyroid carcinoma cell lines. Our experiments clearly show that overexpression of MDR1, MRP or LRP is sufficient to confer anti-cancer drug resistance. Also, there was no mutation at amino acids 439, 450 and 803 in KBG-2 and CA500, expressing MDR1 and MRP, respectively (data not shown, [17,22]). On the other hand, Bugg et al. have identified a mutation in a human cell line selected for resistance to teniposide [3]. Subsequently, Danks et al. have demonstrated by single-strand conformation polymorphism analysis that a second mutation is present in teniposide-resistant cell lines [4]. It would be interesting to examine the relationship between teniposide resistance and mutations in DNA topoisomerase IIa. Our cell lines are resistant to etoposide to varying degrees. Therefore, it is quite difficult to demonstrate whether the observed mutations are actually responsible for drug resistance It has been reported that introducing either of the mutations, Arg450Gln or Pro803Ser into the VM-1 cell line results in an enzyme that can confer drug resistance to yeast [9]. However, these experiments cannot exclude the possibility that other mechanisms of drug resistance are also present in the mammalian cell lines from which the mutations were first identified. Our

present experimental evidence is analogous to previous experiments showing that overexpression of MDR1 and MRP is sufficient to confer anti-cancer drug resistance [7]. It will be necessary to examine the presence or absence of DNA topoisomerase IIa mutations in many multidrug-resistant cell lines available worldwide in order to calculate the significance of DNA topoisomerase IIa gene mutation in multidrug resistance.

Acknowledgements The authors thank Prof. T. Andoh, Department of Engineering, Soka University, for his fruitful discussion.

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