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DETECTION OF TOPOISOMERASE I GENE POINT MUTATION IN CPT-11 RESISTANT LUNG CANCER CELL LINE Naohiro

Kubotal>z, Fumihiko Kanzawa 1, Kazuto Nishiol , Yuichirou Takedal, Tohru Ohmoril, Yasuhiro Fujiwaral, Yoshiteru Terashimaz and Nagahiro Saijol

‘Pharmacology 2Department

Received

National Cancer Center Research Division, Tsukiji 5-1-1, Chuo-ku, Tokyo 104, Japan

of Gynecology, Jikei Medical College, Minato-ku, Tokyo, Japan

September

3,

Institute,

Nishi-Shinbashi

3-25-8,

1992

SUMMARY: CPT-11, a recently developed topoisomerase I (Topo I) inhibitor, attracts the attention not only of basic researchers but also of clinicians because of its high antitumor activity. The CPT- 11 resistant human lung cancer cell line, PC-7/CPT, showed IO-fold resistance compared to parental cell line, PC-7. The total activity of Topo I in the resistant cell line was one fourth that of the parental sensitive cell line. The Topo I from the resistant cells was also 5-fold more resistant to the inhibitory effect of CPT-11 than that of the parental cells. We speculated that the alteration of the Topo I gene may be responsible for the change in topoisomerase activity of the CPT-11 resistant cell line. Therefore, we analyzed the mutation of Topo I gene using the method of single strand conformation polymorphism of polymerase chain reaction and the reverse into ten fragments which transcriptase. We divided Topo I cDNA overlapped each other and covered whole coding sequences of the Topo I cDNA. We observed mobility shift of two fragments in the PC-7/CPT, suggesting the presence of some mutations in these fragments. We performed the direct-sequencing of these portions by the dideoxy chain termination method and observed an altered sequence having a G to A This base substitution results in replacement of base change in PC-7/CPT. These results the conserved threonine at 729 position with alanine. suggest that the point mutation of Topo I gene is related to the decreases of Topo I activity and the sensitivity to Topo I inhibitor in PC-7/CPT cells. 0 1992 Academic

Pres*,

Inc.

ABBREVIATIONS: Topo I, topoisomerase I; Topo II, topoisomerase II; CPT- 11, 7-ethyl-lo-(4-( 1-piperdino)I-piperidino) carbonyloxy camptothecin; SSCP, single strand conformation polymorphism; PCR, acid. polymerase chain reaction; EDTA, ethylenediaminetetraacetic 0006-291X/92 571

$4.00

#Copyright 0 1992 by Academic Press, Inc. All r,‘ghts of reproduction in any form reserved.

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Camptothecin is a specific inhibitor of mammalian DNA topoisomerase I (Topo I) which catalyzes and changes the topological state 7-ethyl- 1Oof duplex DNA by single strand breakage-resealing cycles (1). (4-( 1-piperdino)-1 -piperidino) carbonyloxy camptothecin, CPT- 11, developed in Japan has wide spectrum antitumor activity against human We have solid tumors such as colon, stomach and lung cancers (2-5). established CPT-11 resistant cell line (PC-7/CPT) from a human non-small PC-7/CPT exhibited about lo-fold cell lung cancer cell line (PC-7) (6). increase in resistance to CPT-11. Topo I activity was decreased in PC7/CPT compared to parental cells by the DNA relaxation assay. Topo I from the resistant cells was shown to be 5-fold more resistant to the inhibitory effect of CPT-11 than that from the parental cells. From these results, we speculated that the some alteration of Topo I might cause the change in Topo I activity of the CPT-11 resistant cell line. Recently, Sekiya et al. developed the rapid and sensitive method for the detection of minor sequence variations in DNA fragments, namely single strand conformation polymorphism (SSCP) analysis of polymerase The present study was designed to chain reaction (PCR) products (7,8). see if Topo I gene mutation could be responsible for resistance of the PC7/CPT cells. MATERIALS

& METHODS

Cell line and Culture: Two human tumor cell lines and their CPT-11 resistant sublines were used in the present study. PC-7, derived from a patient with pulmonary adenocarcinoma, was provided by Dr. Y Hayata of the Tokyo Medical College. PC-7/CPT is a subline resistant to CPT-11 as reported previously (6). The RPM1 8402 and CPT-K-5 cell lines, kindly donated by Dr. T Andoh of the Aichi Cancer Research Institute, are T-cellderived acute lymphoblastic leukemia cell line and its CPT-11 resistant All the cell lines were propagated in RPM1 subline, respectively (9,10). 1640 medium supplemented with 10% heat-inactivated fetal bovine (FBS) (Gibco, Tokyo) plus penicillin (100 units/ml) and serum streptomycin (100 ug /ml) (RPMI-FBS) at 37 “C in a humidified incubator with an atmosphere of 5% CO2. RNA extraction and reverse transcriptase reaction: Total cellar RNA was extracted from the parent and CPT-1 l-resistant cell lines by the acid guanidine thiocyanate-phenol-chloroform extraction method (11). Samples of total cellular RNA (1 ug) were annealed with synthetic short DNA of 20 nucleotides, primer 53 (as shown in Table l), which was complementary to the 3’ noncoding region of the Topo I mRNA. After that, mixtures were transcribed with M-MLV reverse transcriptase (Betheda Research Laboratories) in the reaction mixture (14 ~1) containing ribonuclase inhibitor, RNasin as described previously (12). PCR-SSCP analysis: Oligonucleotide primers were synthesized by the phosphoroamidite method with a 391 DNA synthesizer and purified on 572

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A 269 --

AND

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E

G

I

257

376

4%

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bp

---

361

361

236

266

366 bp

Fig. 1. Regions of topoisomerase I cDNA amplified and subjected to SSCP analysis. The coding region of topoisomerase I cDNA were divided into ten fragments (A to J). Their positions are indicated by a bar with arrows at both ends, their nucleotide lengths are indicated under .or over the bars. The coding region is indicated by an open box. ATG and TAG indicate the initiation and termination codons, respectively, of the reading frame of the topoisomerase I cDNA.

OPC columns (Applied Biosystems, CA). The names and nucleotide sequences of the primers used and names and the length of the amplified DNA fragments were listed in Table 1. One pl of reverse transcriptase reaction mixture was subjected directly to the polymerase chain reaction to amplify the regions of Topo I cDNA (Fig 1) in the mixture (10 ~1) as described (13), using a pair of appropriate oligonucleotides as primers. The 5’ ends of these primers were labeled with [@PI ATP by the polynucleotide kinase reaction as described previously (14). The resultant products of the PCR were diluted with 30 to 50 volumes of loading solution containing 90% formamide, 20 mM ethylenediaminetetraacetic acid (EDTA), and 0.05% xylen cyan01 and bromophenol blue, denatured at 80 OC, and applied (1 pi/lane) to 6% polyacrylamide gel containing 90 mM Tris-borate (pH 8.3) and 4 mM EDTA, with or without 10% glycerol as specified. Electrophoresis was performed at 30W for 2 to 6 h under cooling with a fan. The gel was dried film (Amersham, Sweden) on filter paper and exposed to Hyperfilm-MP at -80 “C for 0.5 to 12 h with an intensifying screen (Lightning Plus; DuPont Cronex). Direct DNA sequencing: Nucleotide sequences were determined by the dideoxy chain termination method with asymmetric PCR products (15). Fifty cycles of the asymmetric PCR were performed as described with an uneven molar ratio (10 to 1) of the primers (15). After dilution, the amplified reaction mixtures were deionized in a micro-concentrator, Centricon 30 (Amicon, MA) and the single stranded DNAs were annealed to a 5’-labeled primer. Chain elongation and termination were performed with Sequenase TM kit (United States Biochem.) and the products were analyzed by electrophoresis in 6% polyacrylamide gel containing 7M urea. RESULTS SSCP analysis: Regions of the human Topo I cDNA were analyzed by PCR-SSCP. Ten fragments of the Topo I such as A, B, C, D, E, F, G, H, I and J were amplified by PCR. These ten fragments overlapped each other and 573

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Table 1. Primers used for amplification of the topoisomerase Name A

Amplified Codons included 1-89

fragment Length

I cDNA

Primers (bp)

Name

269

Al A2 Bl 82 Cl

B

71-191

361

C

138-225

263

D

209-309

301

E

269-354

257

F

340-418

236

G

403-528

378

Gi G2

H

515-611

289

I

598-744

439

J

684-765

365

Hl H2 I1 I2 Jl J2 J3

c2 Dl D2 El E2 Fl F2

Sequence S 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’

GACATGAGTGGGGACCACCT CGll-fl-TCCl-TGTCTCTGTC ATGGAAGCTCAGAAAAGCAT TTGTTATCTGGCTCAGGAAC AGACCTCGAGATGAGGATGA GGACCT-I-TATGTTCTAGGAA AGCGCTATCCTGAAGGCATC AAATACTGGCTCATCTGGGT ACTACCAAGGAAATATTTAG TTGAAGTTAGCAATCCTCTC TCTGTAT-TATGGATAACCAC CTCTGTCCAGGAAACCAGCC GAAGTCCGGCATGATAACAA GTCAAACTCTACCACATAT-K ACCCAGAG-ITGGATGGTCAG T-KTCATCCGGGGCTGTCAG TACAGCAGCAGCTAAAAGAA TAAATCTTCTCAATTGGGAC GTAGAGTCAAAGAAGAAGGC Gll-TGlTAAGACTTGCTGCC TTATAATATCTCCAGGTTTA

3 3 3 3 3 3’ 3 3 3’ 3 3’ 3 3’ 3 3 3 3 3’ 3 3 3

covered whole coding sequences of Topo I cDNA of about 2.3 kilobase pairs. The primers used for amplification of the Topo I cDNA were shown they were subjected to SSCP analysis. in Table 1. After amplification, Andoh et al. reported that sequence differences of Topo I cDNA, which comigrated with fragment H used in the present study, between RPM18402 and CPT-K-5 cells (16). Therefore, we used these cells as positive controls. The mutations of Topo I of RPM1 8402 and CPT-K-5 were confirmed as mobility shifts in fragment H by SSCP analysis (Fig. 2). These correspond to the mutation of codons 583 and 533. These results suggested that our PCR-SSCP could detect the mutation of Topo I cDNA. Then, we conducted PCR-SSCP of these fragments in PC-7 and PC-7/CPT. Results were summarized in Fig. 2 and 3. Although no mobility shift was observed in fragment A, B, C, D, E, F, G and H in PC-‘IICPT, we could detect the mobility shift in fragment I and J for PC-7/CPT (Fig. 3). Fragment I and J corresponded with the codons 598-744 and 684-765, respectively (Fig. 1). From this data, we concluded that some mutations were present in codon 598-765 of Topo I cDNA from PC-7/CPT cells. Direct DNA sequencing: Nucleotide sequences of cDNA fragments I and J were determined by the dideoxy chain termination method on asymmetric PCR products. Difference between the PC-7 and the PC-7/CPT cDNA sequences was one nucleotide substitution changing Thr 729 &CA) 574

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FRAGMENT

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FRAGMENT

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E

12345

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C

12345

FRAGMENT

F

FRAGMENT

D

12345

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Fig. 2. RT-PCR-SSCP analysis of fragment A to H in the topoisomerase I cDNA. Total cellar RNA from four tumor cell lines (PC-7, PC-7/CPT, RPM18402, CPT-KS; lane 1 to 4, respectively) and human placenta (lane 5) was subjected to RT-PCR-SSCP analysis. Electrophoresis was performed in 6% polyacrylamide gel containing with 10% glycerol at 30W for 2 to 4 h under a cooling fan.

of PC-7

I to Ala (GCA) in Fig. 4 (numbered according to D’arpa et al. (17) ). Because codon 729 was overlapped region of fragment I and J (Fig. l), this base change was consistent with the mobility shifts of fragment I and J at SSCP analysis (Fig. 3). topoisomerase

DISCUSSION We have examined the point mutation of Topo I gene in a CPTresistant lung cancer cell line, PC-7/CPT using PCR-SSCP and following direct DNA sequencing, suggesting a point mutation in Topo I in CPT-11 Topo I cDNA from PC-7/CPT contained an altered resistant cell lines. This base substitution results in sequence having a G to A base change. the replacement of the threonine at 729 position with an alanine in C terminal of the protein. In the topoisomerase II (Topo II) inhibitor resistant cell lines, different position of mutation in the Topo II gene have been reported. However, the site of mutations was very close each other and the ATP-binding domain fold of Topo II (18, 19). corresponding Futhermore, Lee et al. have reported that the codon 486 of Topo II is the specific

position

for

resistance

to

amsacrine

in

the

different

experiment

(20). Andoh et al. have reported point mutation of Topo I gene from CPT11 resistant cells (9, 16). The point mutation Topo I gene from PC-7/CPT, 575

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J

12345

729

Thr

03

729

Ala

04

Fig. 3. R-I‘-PCR-SSCP ana lysis of fragment I and J in the Total cellar RNA from four tumor cell lines (PC-7, topoisomerase I cDNA. PC-7/CPT, RPM18402, CPT-KS; lane 1 to 4, respectively) and human placenta (lane 5) was subjected to RT-PCR-SSCP analysis. Electrophoresis was performed in 6% polyacrylamide gel containing 10% glycerol at 30W for 4 h under a cooling fan. Fig. 4. Nucleotide sequence of fragment J of topoisomerase I cDNA. Samples of 1 l,tg of total cellar RNA from PC-7 and PC-7/CPT were reverse transcribed and the cDNAs obtained were amplified by the PCR. The PCR products were separated by electrophoresis in 6% polyacrylamide gel. Fragment J of 365 bp was eluted from the gel and subjected to nucleotide sequencing by the dideoxy chain termination method. in a different site as compared with presented here, was demonstrated Andoh’s resistant cell line. Considering these results, the mutation portion might be variable in different CPT-11 resistant cells lines. We previously demonstrated that the decreased Topo I activity contributed to resistance to CPT-11 in PC-7/CPT (6). Immunoprecipitation by polyclonal anti-Top0 I antibody showed that no difference in Topo I level was observed in PC-7 and PC-7/CPT (data not shown). This meant that the amount of Topo I protein was not related to the decreased activity of Topo I in PC-7/CPT. Therefore, the point mutation in codon 729 might be related to the decreased Topo I activity in PC-7/CPT. To elucidate the relationship between this mutation and activity, we have to conduct site-directed mutagenesis. In this study we developed the suitable condition for the detection of alteration of Topo I gene using PCR-SSCP. This methods is rapid and sensitive to analyze the gene mutation using small samples of 1 ug. Thus this method is useful for the analysis of clinical material. 576

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ACKNOWLEDGMENTS : This work was supported by a Grant-in-aid for Cancer Research from the Comprehensive Ten-year Strategy for Cancer Control, from the Ministry of Health and Welfare, JAPAN. We thank Dr. Andoh for kindly providing us RPM18402 and CPT-KS cell lines. We would

like

to

thank

Dr.

Awtar

Krishan

for

his

critical

review

of

this

manuscript. Dr. Awtar Krishan’s visit was supported by the Visiting Scientist Program of the Foundation for Promotion of Cancer Research based on the Comprehensive lo-year Strategy for Cancer Control. N. K. and T. 0. are awardees of a Research Resident Fellowship from the Foundation

for Promotion

of Cancer

Research,

Japan.

REFERENCES 1. Thomsen, B., Mollerup, S., Bonven, B.J., Blocker, H., Nielsen, O.F., and Westergaard, 0. (1987) EMBO 5.6, 1817-1823. 2. Takeuchi, S., Takamizawa, H., Takeda, Y., Okawa, T., Tamaya, T., Noda, K., Sugawa, T., Sekiba, K., Yakushiji, M., and Taguchi, T. (1991) Proc. ASCOlO, 189.(Abstract) 3. Fukuoka, M., Niitani, H., Suzuki, A., Motomiya, M., Hasegawa, K., Nishiwaki, Y., Kuriyama, T., Ariyoshi, Y., Negoro, S., Masuda, N., Nakajima, S., and Taguchi, T. (1992) J. Clin. Onco1.10, 16-20. 4. Simada, Y., Yoshida, M., Wakui, A., Nakao, I., Futatsuki, K., Sakata, Y., Kambe, M., and Taguchi, T. (1991) Proc. ASCOlO, 135.(Abstract) 5. Ohe, Y., Sasaki, Y., Shinkai, T., Eguchi, T., Tamura, T., Kojima, A., Kunikane, H,. Okamoto, H., Karato, A., Ohamatsu, H., Kanzawa, F., Saijo, N. (1992) J Nat1 Cancer Inst 84, 972-974. 6. Kanzawa, F., Sugimoto, Y., Minato, K., Kasahara, K., Bungo, M., Nakagawa, K., Fujiwara, Y., Liu, L.F., and Saijo, N. (1990) Cancer Res.50, 5919-5924 7. Orita, M., Iwahana, H., Kanazawa, H., Hayashi, K., and Sekiya, T. (1989) Proc. Natl. Acid. Sci. USA. 86, 2766-2770. 8. Orita, M., Suzuki, Y., Sekiya, T., and Hayashi, K. (1989) GenomicsS, 874-879. 9. Andoh, T., Ishii, K., Suzuki, Y., Ikegami, Y., Kusunoki, Y., Takemoto, Y., and Okada, K. (1987) Proc. Natl. Acid. Sci. USA. 84, 5565-5569. 10. Minowada, J. and Moore, G.E. (1975) Comparative Leukemia Research 1973: Leukemogenesis, p. 251. Univ. of Tokyo Press, Tokyo. 11. Chomczynski, P. and Sacchi, N. (1986) Anal. Biochem.162, 156-159. 12. Kawasaki, E.S., Clark, S.S., Coyne, M.Y., Smith, S.D., Champlin, R., Witte, O.N., and Mccormick, F.P. (1988) Proc. Natl. Acid. Sci. USA. 85, 5698- 5702. 13. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T.. Mullis, K.B., and Erlich, H.A. (1988) Science239, 487-491. 14. Hayashi, K., O&a, M., Suzuki, Y., and Sekiya, T. (1989) Nucleic Acids Res. 17, 3605. 15. Gyllensten, U.B. and Erlich, H.A. (1987) Proc. Natl. Acid. Sci. USA. 84, 9059-9063. 16. Tamura, H., Kohchi, C., Yamada, R., Ikeda, T., Koiwai, O., Patterson, E., Keene, J.D., Okada, K., Kjeldsen, E., Nishikawa, K., and Andoh, T. (1990) Nucleic Acids Res.19, 69-75. 17. D’Arpa, P., Machlin, P.S., Ratrie III, H., Rothfield, N.F., Cleveland, D.W., and Eamshaw, W.C. (1988) Proc. Natl. Acid. Sci. USA. 85, 2543-2547. 18. Hinds, M., Deisseroth, K., Mayers, J., Altschuler, E., Jansen, R., Ledley, F.D., and Zwelhng, L.A. (1991) Cancer Res.51, 4729-4731. 19. Bugg, B.B., Danks, M.D., Beck, W.T., and Suttle, D.P. (1991) Proc. Natl. Acid. Sci. USA. 88, 7654-7658. 20. Lee, M.S., Wang, J.C., and Beran, M. (1992) J. Mol. Bio1.223, 837-843. 577