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Induction of λ-glutamylcysteine synthetase gene expression by platinum drugs in peripheral mononuclear cells of lung cancer patients
Annals of Oncology 10: 455^(60, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands.
Original article Induction of y-glutamylcysteine synthetase gene expression by platinum drugs in peripheral mononuclear cells of lung cancer patients T. Oguri,1 Y. Fujiwara,1'2 M. Miyazaki,1 T. Takahashi,1 T. Kurata,1 M. Yokozaki,1 N. Ohashi,1 T. Isobe,1 O. Katoh3 & M. Yamakido1 {
Summary
Results: There were no differences in the baseline expression levels between normal volunteers and lung cancer patients in any of the genes. After platinum drug administration, the heavy subunit of y-GCS (y-GCSh) expression level increased 2.5-fold within 24 hours and the increase persisted for a month, whereas the light subunit of y-GCS (y-GCSl) expression level did not show an early response but had increased after a month. By contrast, the MRP, cMOAT and topo I expression levels were similar before, during and after chemotherapy. Conclusions: These results suggest that the gene expression levels of both subunits of y-GCS play an important in vivo role in platinum drug resistance.
Background: To investigate in vivo the roles of Y-glutamylcysteine synthetase (y-GCS), multidrug resistance-associated protein (MRP), human canalicular multispecific organic anion transporter (cMOAT) and DNA topoisomerase I (topo I) in relation to platinum drug resistance, we monitored the changes of the steady-state levels of the mRNAs for these factors in peripheral mononuclear cells (PMN) after completing platinum drug administration. Patients and methods' PMN from 46 subjects were studied. We obtained PMN from 14 previously untreated lung cancer patients and 14 normal volunteers to measure the baseline gene expression levels. We then obtained PMN from 18 patients with previously untreated advanced lung cancer before and after they received platinum drug treatment. We analyzed the gene expression levels by using the quantitative reverse transcription Key words: cMOAT, y-GCS heavy subunit, y-GCS light subpolymerase chain reaction (RT-PCR). unit, MRP, platinum drug resistance
Introduction It is important to clarify the underlying mechanism of resistance to platinum drugs. Several mechanisms, including increased intracellular drug detoxification and reduced drug accumulation, have been suggested. The former is supported by reports of increased levels of glutathione (GSH) and of the rate-limiting enzyme of GSH de novo synthesis, y-glutamylcysteine synthetase (y-GCS) [1-5], and the latter may involve the ATPdependent GSH S-conjugate export (GS-X) pump [3-9]. Several investigators have demonstrated that multidrug resistance-associated protein (MRP) and human canalicular multispecific organic anion transporter (cMOAT) serve GS-X pump activity [10-14], but whether either MRP or cMOAT is identical to the efflux pump for the GSH-platinum complex remains uncertain. We previously demonstrated that the expression levels in autopsy specimens of the y-GCS and MRP genes, but not those of the cMOAT and topoisomerase I (Topo I) genes, are associated with platinum drug exposure, and we have also reported preliminary results showing that
the expression levels of y-GCS heavy subunit (y-GCSh) in peripheral mononuclear cells (PMN) of eight lung cancer patients increased after platinum drug administration [15]. However, whether the y-GCS expression level in PMN can be used as a marker of resistance to platinum drugs, and whether it is affected over a long period by platinum drugs, remains to be determined. Therefore, in the present study we examined the in vivo induction of mRNAs for both of the subunits of y-GCS, MRP, cMOAT and Topo I to clarify their roles in platinum drug resistance.
Patients and methods Cell lines and cell culture Human lung adenocarcinoma (PC-9) cells were maintained in a humidified chamber (37 °C, 5% CO 2 ) in RPMI 1640 medium (Nissui CO., Tokyo, Japan) supplemented with 10% heat-inactivated fetal calf serum (Mitsubishi Kasei Co., Tokyo Japan), penicillin (100 U/ml) and streptomycin (100 ug/ml). For the induction experiments parental cells in the late-log phase were harvested and seeded into 10 cm 2 tissue
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Second Department of Internal Medicine, Hiroshima University School of Medicine; 2 Evaluation Division I, Pharmaceuticals and Medical Devices Evaluation Center, National Institute of Health Sciences; 3 Department of Environment and Mutation, Research Institute for Radiation Biology and Medicine, Hiroshima University, Japan
456 Table I. The PCR condition and the sequence of primers. Gene
Forward primer
Backward primer
Annealing (°C) Cycles
GCSh GCS1 MRP cMOAT Topo I P-actin
5'-AGGCCAGATACCTTTATGATCAGT-3' 5'-TGTCTTGGAATGCACTGTATCTCATGC-3' 5'-TGGGACTGGAATGTCACG-3' 5-CTAATCTAGCCTACTCCTGC-3' 5'-GGAGAGACCTGAAAAGTGCTAA-3' 5'-AAGAGAGGCATCCTCACCCT-3'
5'-GCTGTCTATTGAGTCATATCGGGATTTAC-3' 5' •TTCAATAGGAGGTGAAGCAATGATCAC-3' 5' •AGGAATATGCCCCGACTTC-3' 5'-CTGCAGCTCTCTCTTCATGTGC-3' 5'-TAAATCTTCTCAATTGGGAC-3' 5'-TACATGGCTGGGGTGTTGAA-3'
64 64 60 64 59 55
annealing at 55-64 °C for 30 s and extension at 72 °C for 1 min, followed by afinalincubation at 72 °C for 7 min. The PCR conditions and the sequences of the y-GCSh, y-GCSI, MRP, cMOAT, Topo 1 and P-actin primers are presented in Table 1. The PCR products were 221, 196, 293, 275, 247 and 218 base pairs (bp) long, corresponding to y-GCSh, y-GCSI, MRP, cMOAT, Topo I and P-actin cDNA, respectively.
Patients and isolation of PMN PMN from 46 subjects were studied. We obtained PMN from 32 lung cancer patients who were admitted to Hiroshima University Hospital from March 1996 to September 1997, after written informed consent had been obtained. None of the patients had previously received chemotherapy. We also obtained PMN from 14 normal volunteers after obtaining their written informed consent. Hepannized blood samples (5 ml) were taken just before the treatment (0 hours) and six hours, 24 hours and 28 days after the last dose of the platinum drug administered. The PMN were separated immediately using a lymphocyte preparation medium (Lymphoprep®, NYCOMED PHARMA, AS, Oslo, Norway) and washed with cold calcium-free and magnesiumfree Dulbecco's phosphate-buffered saline (Nissui, Tokyo, Japan). Contaminating erythrocytes were lysed with an NH4C1 solution and the cells were centrifuged at 5000 rpm for 5 mm. The pellets were used for RNA extraction or stored at -80 °C until analysis.
Quantitation of PCR products and analysis of mRNA expression
The PCR products were electrophoresed on 2% (w/v) agarose gels, transferred to nylon membranes (Hybond N+; Amersham) and detected by hybridization with 32P-labeled cDNA probes. After each filter was washed the radioactivity level was measured with a laser imaging analyzer (BAS-2000; Fuji Photo Film, Tokyo, Japan). We used y-GCSh, y-GCSI, MRP, cMOAT, and P-actin oligonucleotides as the probes as described previously [15]. We used topo I oligonucleotides as the probe and the primers were: forward 5'-GAAGTCCGGCATGATAACAA-3' and reverse S'-GTTTGTTAAGACTTGCTGCC-S'. The topo I PCR products were 1208 bp long. In each sample the radioactivity associated with the gene expression level was expressed relative to the p-actin expression level.
Statistical analysis RNA extraction and reverse transcription Total cellular RNA was extracted using the guanidinium isothiocyanate-phenol method and cDNA was synthesized using random hexamer primers (Amersham, Buckinghamshire, UK) with Superscript RNase H - reverse transcriptase (GIBCO-BRL, Bethesda, MD, USA) as described previously [16].
Northern blot hybridization Northern blots of total cellular RNA extracted from the lung cancer cell lines were hybridized with cDNA probes for both subunits of y-GCS as described previously [1]. We used y-GCSh and y-GCS light subunit (y-GCSl) oligonucleotides as the probes as described previously [15].
Contingency table analyses based on x2 statistics were used to determine the significance of associations between categorical variables Differences in expression level between two groups of PMN were analyzed using the Mann-Whitney U-test or the Wilcoxon signedranks test. All of the gene expression levels were skewed toward higher expression levels, and were subjected to logarithmic transformation, so that they more closely approximated a normal distribution, before a parametric test (repeated measures of ANOVA) was performed. The statistical calculations and tests were performed using Stat View J4.ll Software (ABACUS Co., CA, USA) and a Macintosh computer. All of the statistical tests were two-sided, the data are expressed as medians and ranges, and differences with /"-values of less than 0.05 were considered significant.
Results Polymerase chain reaction The reverse-transcribed cDNA from each sample was amplified by the polymerase chain reaction (PCR) using primers based on the y-GCSh. y-GCSl, MRP, cMOAT. Topo I and p-actin (internal control) gene sequences. After pre-denaturation at 94 "C for 5 min, the cDNA was added to 5 ul of the PCR mixture: 1 ul 10x PCR buffer (100 mM Tris-HCl, pH 9.0, 500 mM KC1), I ul 15 mM MgCl2, 2 ul distilled water, 0.2 ul 20 mM dNTPs (Takara, Tokyo, Japan). 0.2 ul 50 uM forward primer, 0.2 ul 50 uM reverse primer, and 0.4 ul (0.2 u) Taq polymerase (Promega. Madison. WI. USA). Amplification was carried out using a thermal cycler (Geneamp PCR System 2400; Perkin Elmer Applied Biosystems Division. Norwalk. CO, USA). Each amplification cycle for the reactions comprised denaturation at 94 ~C for 30 s,
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culture flasks (Becton Dickinson Labware, NJ, USA) at a density of 1 x 106 cells/ml. After a 1-hour preincubation period the cells were incubated with 5 ug/ml of cisplatin (CDDP) for one, four, eight and 24 hours. The CDDP concentration was chosen as described previously [1]. CDDP was obtained from the Bristol Myers Co. (Tokyo, Japan).
22 22 24 24 24 20
First, to investigate the baseline expression levels of the genes encoding the subunits of y-GCS, MRP, cMOAT and Topo I in PMN, we compared the expression levels of 14 normal volunteers with those of 32 lung cancer patients who had not been treated with any chemotherapy before this study. Ten of the normal volunteers were male and four female, they were aged from 24 to 37 years (median of 29 years), and six of them were smokers. The lung cancer patients included 25 males and seven females, with an age range of 41 to 82 years (median of 67 years), and almost all (28 of 32) were smokers (Table 2).
457 Table 2. Patient characteristics."
Table 3. Comparison of baseline gene expression levels in normal volunteers to those in lung cancer patients. b
Pt Age Sex Smoker Histol- TNM PS Therapy no. (years) ogy stage
68 82 79 58 59 62 50 59 68 51 69 73 73 76 67 69 62 41 66 76 67 54 44 68 71 61 62 75 59 72 51 65
M M F F M M M M F M M M M F M M M M M F M M M M M M M M M M F F
AD SQ AD AD AD AD AD AD SQ SQ AD AD SQ AD SM AD SM SM SM AD SM SQ AD AD SQ AD SQ SQ AD AD SQ AD
IV
Ilia IV IV 1Mb
Ilia I IV IHb IV IV I IHb I IHb IV
Ilia IHb IV IV IHb IHb 1Mb IV IV IV IV IV IV IV IV IV
0 2 0 4 2 0 0 2 0 0 3 0 0 0 0 0 0 3 1 2 0 0 0 0 0 0 0 0 0 0 0 0
SM5887 BSC BSC BSC RT
Operation Operation BSC
SM5887 SM5887 BSC
Operation BSC
Operation CDDP + CPT-11 CDDP + CPT-11 CDDP + CPT-11 CDDP+VP CBDCA + VP CBDCA CBDCA + VP CDDP + CPT-11 CDDP + CPT-11 CBDCA CBDCA CBDCA CBDCA CBDCA CBDCA CBDCA CBDCA CBDCA
64 50 55 94 332 341 341 87 101 341 394 331 284 296 459 375 459 371
* Blood samples from patients number 1 — 14 were obtained just before each therapy. b SM5887, newdrugofanthracyclin. c Platinum dose which was given during the study period. Abbreviations: M - male; F - female. + patient who had smoked; - patient who had never smoked; SM - small cell carcinoma; AD - adenocarcinoma; SQ squamous cell carcinoma; PS - performance status; BSC - best supportive care; RT - irradiation; CDDP - cisplatin; CPT-11 - irinotecan hydrochloride; CBDCA - carboplatin; VP - etoposide.
There were no differences in the baseline expression levels between the normal volunteers and the lung cancer patients in any of the genes tested (Table 3, MannWhitney U-test). To examine whether y-GCSh and y-GCSl mRNAs are induced in lung cancer cells in response to platinum drugs we exposed PC-9 cells to CDDP before Northern blot analysis. We found that the y-GCSh mRNA levels had increased about three-fold within 24 hours after exposure to CDDP, whereas the y-GCSl mRNA had not changed (Figure 1). These data were used for determining the sampling time points in the next study. We then monitored the expression level of each gene in PMN from 18 advanced-lung-cancer patients who were treated with a platinum drug only or by platinum drug-based chemotherapy (presented in Table 2, patient numbers 15-32). Fifteen of the 18 patients were male and three female, ranging in age from 41 to 76 years old, with a median of 66 years, and almost all of them (15 of 18) were smokers. CDDP and carboplatin (CBDCA) were administered as a one-hour infusion on day 1. The
Gene expression levels
GCSh GCSI MRP cMOAT Topol
Normal volunteers
Lung cancer patients
0.124(0.054-0.225) 0.135(0.036-0.330) 0.241 (0.040-0.487) 0.260(0.096-1.045) 0.399(0.171-0.638)
0.136(0.011-0.262) 0.177(0.011-0.656) 0.196(0.022-0.710) 0.180(0.038-0.950) 0.388 (0.118-1.225)a
The data was expressed as the median (range). a Median (range) of 28 patients; 4 patients (patient numbers 29-32 in Table 2) were not examined.
PC-9 C 1h 4h 8h 24h /-GCSh
4.1Kb 3.2Kb 4.1Kb
Y-GCSl
1.4Kb 28S Figure 1. Expression of y-GCSh and y-GCSl mRNA in PC-9 cells and in PC-9 cells that had been exposed to CDDP, detected by Northern blot analysis. 28S ribosomal RNAs stained by ethidium bromide were used as an internal control. C - cells not exposed to CDDP; 1 —24 h — cells incubated with 5 ug/ml CDDP for 1, 4, 8 and 24 hours.
CDDP dose was 80 mg/m2 and the CBDCA dose was calculated using Calvert's formula: dose (mg/body) = target AUC x (GFR + 25) [17, 18] and we set the target AUC at 7 mg/ml x min [19]. As shown in Table 4, the y-GCSh expression level increased immediately after exposure to a platinum drug (P < 0.0001, repeated measures of ANOVA), but there was no immediate change in the y-GCSl, MRP, cMOATand Topo I expression levels. To examine long-term effects of the platinum drugs we measured the gene expression levels again, 28 days after completion of platinum drug administration. We were able to obtain PMN from only six of the aforementioned 18 patients for the second measurement. Our results demonstrate that mRNA levels of both y-GCSh and y-GCSl were significantly higher 28 days after platinum drug treatment (both P-values were 0.0464, Wilcoxon signed-ranks test, Table 5), although there were still no differences in the MRP, cMOATand Topo I mRNA levels. These results also suggest that the y-GCSh
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
Platinum dose (mg)c
458 Table 4. Gene expression levels within 24 hours after platinum drug administration. Gene
GCSh GCS1 MRP cMOAT Topo Ia
P-value
Gene expression levels 0 hours
6 hours
24 hours
0.096(0.013-0.211) 0.161 (0.011-0.656) 0.187(0.094-0.710) 0.180(0.038-0.575) 0.450(0.118-1.225)
0.166(0.076-0.752) 0.177(0.055-0.588) 0.218(0.050-1.176) 0.174(0.024-0.543) 0.737(0.309-1.297)
0.238 (0.059-0.533) 0.169(0.028-0.719) 0.189(0.046-2.140) 0.190(0.043-0.567) 0.777(0.203-1.378)
< 0.0001 NS NS NS NS
Abbreviation: NS - not significant. The data was expressed as the median (range). P-values of less than 0.05 were considered to be significant using repeated measures of ANOVA. a Topo I expression levels were calculated from 28 patients; 4 patients (patient numbers 29-32 in Table 2) were not examined.
P-value
Gene expression levels
GCSh GCS1 MRP cMOAT Topo I
Before
After 24 hours
After 28 days
0.140(0.038-0.176) 0.120(0.059-0.245) 0.204(0.101-0.710) 0.220 (0.063-0.575) 0.462(0.331-0.820)
0.343(0.176-0.533) 0.151 (0.044-0.351) 0.384(0.044-1.203) 0.209(0.110-0.422) 0.753 (0.203-0.988)
0.340 (0.074-0.375) 0.301 (0.199-0.915) 0.396(0.161-1.145) 0.211(0.107-0.713) 0.627(0 255-1.111)
0.0464 0.0464 NS NS NS
Abbreviation: NS - not significant. The data from six patients (patient numbers 15, 17, 18, 20-22 in Table 2) was expressed as the median (range). P-values of less than 0.05 were considered to be significant using Wilcoxon signed-ranks test.
gene was induced at 24 hours and that the induction continued for 28 days, whereas the y-GCSl gene showed a delayed response. Discussion In this study we demonstrated that the gene expression levels for both subunits of y-GCS had increased after platinum drug exposure, whereas the MRP, cMOATand Topo I had not. The y-GCSh mRNA level had increased within 24 hours and was still high 28 days after platinum drug exposure. This long-term induction can be explained by the fact that platinum remains in tissues for several months to years after the final exposure [20]. A previous comparison of y-GCSh gene expression in cancer cells and those in their CDDP-resistant cells showed that cells with 1.5- and two-fold increases in y-GCSh expression were 2.9 and 7.2 times more resistant to CDDP, respectively [4]. These results indicate that the 2.5-fold increase in y-GCSh expression that was observed in PMN from lung cancer patients may contribute to platinum drug resistance. Moreover, a transfection study showed that y-GCSh overexpression results in an increased GSH content and platinum drug resistance [5]. When considered together with these reports, our finding of increased y-GCSh mRNA levels after platinum drug exposure suggests a difficulty in anticancer chemotherapy since platinum drug administration itself seems to induce GSH-related platinum drug resistance.
We also found that y-GCSh gene expression increased immediately after CDDP exposure in lung cancer cells in vitro, similar to the results in PMN. Previously O'Dwyer [21] reported that y-GCSh gene expression in the colon correlates with that in PMN. Furthermore, y-GCSh gene expression levels in cancer cells may be associated with resistance to platinum drugs in vitro [22]. These and our present findings indicate that y-GCSh gene expression in PMN can be used as a surrogate marker for resistance, or detoxifying activity, to platinum drugs. Unlike the y-GCSh gene, the y-GCSl gene was not rapidly induced, but the y-GCSl mRNA level had increased 28 days after platinum drug exposure. Since y-GCSh exhibits all of the catalytic activity of the enzyme in GSH synthesis as well as feedback inhibition of GSH [23], rapid consumption of GSH to detoxify the platinum may contribute to the rapid induction of y-GCSh without affecting y-GCSl expression. Whereas, y-GCSl plays a regulatory role, modifying the kinetic properties of y-GCSh [24], increased y-GCSl expression may be needed to keep the y-GCSh expression level high for a long period. The effect of irinotecan hydrochloride (CPT-11) or etoposide (VP-16) on gene induction was a significant problem. Because we had not obtained data from patients who received CPT-11 alone or VP-16 alone, we could not actually describe the effects of these drugs on gene expression. However, our reexamination of 10 patients who were administrated CBDCA alone (patient numbers 20, 24—32 in Table 2) showed that the changes in the levels of expression of each gene were almost
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Table 5. Comparison of gene expression levels between before and 28 days after completion of platinum drug administration.
459
To further elucidate the roles of GSH, GSH-related enzyme and the GS-X pump in platinum drug sensitivity or resistance we are currently examining gene expression levels in tissue samples obtained during surgery from patients with lung cancer.
15.
16.
Acknowledgements This study was supported by Grants-in-Aid from the Ministry of Education, Science and Culture and the Ministry of Health and Welfare of Japan, and a grant from Osaka Cancer Research Foundation. We are grateful to Drs. N. Saijo and K. Nishio (Pharmacology Division, National Cancer Center Research Institute) for providing the coding region of cDNA for the heavy subunit of human liver y-GCS and the PC-9 cells.
17.
18.
19.
20.
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identical to the results of the other 8 patients (data not References shown) and to the results obtained when we analyzed data by combining these two patient data sets. We there1. Fujiwara Y. SugimotoY. Kasahara Ket al. Determinants of drug response in a cisplatin-resistant human lung cancer cell line. Jpn J fore believe that platinum drugs did induce y-GCS gene Cancer Res 1990; 81: 527-35. expression levels. 2. Godwin AK, Meister A. O'Dwyer PJ et al. High resistance to The GSH-platinum complex export pump has not cisplatin in human ovarian cancer cell lines is associated with been identified, although both MRP and cMOAT show marked increase of glutathione synthesis. Proc Natl Acad Sci USA 1992; 89: 3070-4. GS-X pump activity [10-14]. In the present study we 3. Hamaguchi K, Godwin AK, Yakushiji M et al. Cross-resistance observed no MRP and cMOAT overexpression in PMN to diverse drugs is associated with primary cisplatin resistance in after platinum drug administration. However, increased ovarian cancer cell lines. Cancer Res 1993; 53: 5225-32. MRP expression has been associated with platinum 4. Goto S,Yoshida K, MorikawaTet al. Augmentation of transport drug exposure in lung cancers [15] and in human leukefor cisplatin-glutathione adduct in cisplatin-resistant cancer cells. mia cells [25]. In addition, recent in vitro studies demonCancer Res 1995; 55: 4297-301. strated that cMOAT gene expression is induced by 5. Kurokawa H, Ishida T, Nishio K et al. y-Glutamylcysteine synthetase gene overexpression results in increased activity of the CDDP in rat hepatocytes [26] and that cMOAT expresATP-dependent glutathione S-conjugate export pump and cisplasion was associated with sensitivity to CDDP in human tin resistance. Biochem Biophys Res Commun 1995; 216: 258-64. hepatic cancer cells [27]. These results indicate that the 6. Ishikawa T. Ali-Osman F Glutathione-associated c/s-diammineGSH-platinum complex export pump differs among dichloroplatinum(II) metabolism and ATP-dependent efflux from organs and/or cancer types. leukemia cells. J Biol Chem 1993; 268: 20116-25. 7. Ishikawa T, Wright CD, Ishizuka H. GS-X pump is functionally Combination chemotherapy including a platinum overexpressed in cis-diamminedichloroplatinum (Il)-resistant drug and Topo I inhibitors has been used recently to human leukemia HL-60 cells and down-regulated by cell differtreat lung cancer [28-30]. Fukuda et al. [31] showed that entiation. J Biol Chem 1994; 269: 29085-93. there is a biological interaction between these agents, 8. Fujii R, Mutoh M, Sumizawa T et al. Adenosine triphosphatedependent transport of leukotriene C4 by membrane vesicles and we have demonstrated previously that the Topo I prepared from cisplatin-resistant human epidermoid carcinoma gene expression levels may partially determine the sensitumor cells. J Natl Cancer Inst 1994; 86. 1781-9. tivity of the cells to CPT-11 [16]. However, our previous 9. Chuman Y, Chen Z-S, Sumizawa T et al. Characterization of the study showed that Topo I gene expression levels were not ATP-dependent LTC4 transporter in cisplatin-resistant human associated with platinum drug exposure in lung cancer KB cells. Biochem Biophys Res Commun 1996; 226: 158-65. [15]. Accordingly we examined the gene expression of 10. Jedlitschky G, Leier I, Buchholz U et al. ATP-dependent transport of glutathione 5-conjugates by multidrug resistance-associated Topo I in the belief that it could be used as a negative protein. Cancer Res 1994; 54: 4833-6. control in relation to platinum drug exposure in this 11. Leier I, Jedlitschky G, Buchholz U et al. The MRP gene encodes study. On the other hand, another purpose of the measan ATP-dependent export pump for leukotriene C4 and structururement of Topo I gene expression in this study was to ally related conjugates. J Biol Chem 1994; 269: 27808-10. examine whether Topo I gene expression was induced 12. Muller M, Meijer C, Zaman GJR et al. Overexpression of the rapidly after CPT-11 administration. Then we reanalyzed gene encoding the multidrug resistance-associated protein results in increased ATP-dependent glutathione S-conjugate transport. the change in the Topo I gene expression levels in five Proc Natl Acad Sci USA 1994; 91: 13033-7. patients (patient numbers 15-17, 22, 23 in Table 2) who Biicher M, Konig J, Brom M et al. cDNA cloning of the hepatoreceived CDDP and CPT-11, but no Topo I gene induc- 13. cyte canalicular isoform of the multidrug resistance protein, cMrp, tion was observed (data not shown). Therefore, it may be reveals a novel conjugate export pump deficient in hyperbilirubipossible that the gene expression levels of Topo I were nemic mutant rats. J Biol Chem 1996; 271; 15091-8. not induced by platinum drugs and Topo I inhibitors. 14. Madon J, Eckhardt U, Gerloff Tet al. Functional expression of
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Original article Induction of y-glutamylcysteine synthetase gene expression by platinum drugs in peripheral mononuclear cells of lung cancer patients T. Oguri,1 Y. Fujiwara,1'2 M. Miyazaki,1 T. Takahashi,1 T. Kurata,1 M. Yokozaki,1 N. Ohashi,1 T. Isobe,1 O. Katoh3 & M. Yamakido1 {
Summary
Results: There were no differences in the baseline expression levels between normal volunteers and lung cancer patients in any of the genes. After platinum drug administration, the heavy subunit of y-GCS (y-GCSh) expression level increased 2.5-fold within 24 hours and the increase persisted for a month, whereas the light subunit of y-GCS (y-GCSl) expression level did not show an early response but had increased after a month. By contrast, the MRP, cMOAT and topo I expression levels were similar before, during and after chemotherapy. Conclusions: These results suggest that the gene expression levels of both subunits of y-GCS play an important in vivo role in platinum drug resistance.
Background: To investigate in vivo the roles of Y-glutamylcysteine synthetase (y-GCS), multidrug resistance-associated protein (MRP), human canalicular multispecific organic anion transporter (cMOAT) and DNA topoisomerase I (topo I) in relation to platinum drug resistance, we monitored the changes of the steady-state levels of the mRNAs for these factors in peripheral mononuclear cells (PMN) after completing platinum drug administration. Patients and methods' PMN from 46 subjects were studied. We obtained PMN from 14 previously untreated lung cancer patients and 14 normal volunteers to measure the baseline gene expression levels. We then obtained PMN from 18 patients with previously untreated advanced lung cancer before and after they received platinum drug treatment. We analyzed the gene expression levels by using the quantitative reverse transcription Key words: cMOAT, y-GCS heavy subunit, y-GCS light subpolymerase chain reaction (RT-PCR). unit, MRP, platinum drug resistance
Introduction It is important to clarify the underlying mechanism of resistance to platinum drugs. Several mechanisms, including increased intracellular drug detoxification and reduced drug accumulation, have been suggested. The former is supported by reports of increased levels of glutathione (GSH) and of the rate-limiting enzyme of GSH de novo synthesis, y-glutamylcysteine synthetase (y-GCS) [1-5], and the latter may involve the ATPdependent GSH S-conjugate export (GS-X) pump [3-9]. Several investigators have demonstrated that multidrug resistance-associated protein (MRP) and human canalicular multispecific organic anion transporter (cMOAT) serve GS-X pump activity [10-14], but whether either MRP or cMOAT is identical to the efflux pump for the GSH-platinum complex remains uncertain. We previously demonstrated that the expression levels in autopsy specimens of the y-GCS and MRP genes, but not those of the cMOAT and topoisomerase I (Topo I) genes, are associated with platinum drug exposure, and we have also reported preliminary results showing that
the expression levels of y-GCS heavy subunit (y-GCSh) in peripheral mononuclear cells (PMN) of eight lung cancer patients increased after platinum drug administration [15]. However, whether the y-GCS expression level in PMN can be used as a marker of resistance to platinum drugs, and whether it is affected over a long period by platinum drugs, remains to be determined. Therefore, in the present study we examined the in vivo induction of mRNAs for both of the subunits of y-GCS, MRP, cMOAT and Topo I to clarify their roles in platinum drug resistance.
Patients and methods Cell lines and cell culture Human lung adenocarcinoma (PC-9) cells were maintained in a humidified chamber (37 °C, 5% CO 2 ) in RPMI 1640 medium (Nissui CO., Tokyo, Japan) supplemented with 10% heat-inactivated fetal calf serum (Mitsubishi Kasei Co., Tokyo Japan), penicillin (100 U/ml) and streptomycin (100 ug/ml). For the induction experiments parental cells in the late-log phase were harvested and seeded into 10 cm 2 tissue
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Second Department of Internal Medicine, Hiroshima University School of Medicine; 2 Evaluation Division I, Pharmaceuticals and Medical Devices Evaluation Center, National Institute of Health Sciences; 3 Department of Environment and Mutation, Research Institute for Radiation Biology and Medicine, Hiroshima University, Japan
456 Table I. The PCR condition and the sequence of primers. Gene
Forward primer
Backward primer
Annealing (°C) Cycles
GCSh GCS1 MRP cMOAT Topo I P-actin
5'-AGGCCAGATACCTTTATGATCAGT-3' 5'-TGTCTTGGAATGCACTGTATCTCATGC-3' 5'-TGGGACTGGAATGTCACG-3' 5-CTAATCTAGCCTACTCCTGC-3' 5'-GGAGAGACCTGAAAAGTGCTAA-3' 5'-AAGAGAGGCATCCTCACCCT-3'
5'-GCTGTCTATTGAGTCATATCGGGATTTAC-3' 5' •TTCAATAGGAGGTGAAGCAATGATCAC-3' 5' •AGGAATATGCCCCGACTTC-3' 5'-CTGCAGCTCTCTCTTCATGTGC-3' 5'-TAAATCTTCTCAATTGGGAC-3' 5'-TACATGGCTGGGGTGTTGAA-3'
64 64 60 64 59 55
annealing at 55-64 °C for 30 s and extension at 72 °C for 1 min, followed by afinalincubation at 72 °C for 7 min. The PCR conditions and the sequences of the y-GCSh, y-GCSI, MRP, cMOAT, Topo 1 and P-actin primers are presented in Table 1. The PCR products were 221, 196, 293, 275, 247 and 218 base pairs (bp) long, corresponding to y-GCSh, y-GCSI, MRP, cMOAT, Topo I and P-actin cDNA, respectively.
Patients and isolation of PMN PMN from 46 subjects were studied. We obtained PMN from 32 lung cancer patients who were admitted to Hiroshima University Hospital from March 1996 to September 1997, after written informed consent had been obtained. None of the patients had previously received chemotherapy. We also obtained PMN from 14 normal volunteers after obtaining their written informed consent. Hepannized blood samples (5 ml) were taken just before the treatment (0 hours) and six hours, 24 hours and 28 days after the last dose of the platinum drug administered. The PMN were separated immediately using a lymphocyte preparation medium (Lymphoprep®, NYCOMED PHARMA, AS, Oslo, Norway) and washed with cold calcium-free and magnesiumfree Dulbecco's phosphate-buffered saline (Nissui, Tokyo, Japan). Contaminating erythrocytes were lysed with an NH4C1 solution and the cells were centrifuged at 5000 rpm for 5 mm. The pellets were used for RNA extraction or stored at -80 °C until analysis.
Quantitation of PCR products and analysis of mRNA expression
The PCR products were electrophoresed on 2% (w/v) agarose gels, transferred to nylon membranes (Hybond N+; Amersham) and detected by hybridization with 32P-labeled cDNA probes. After each filter was washed the radioactivity level was measured with a laser imaging analyzer (BAS-2000; Fuji Photo Film, Tokyo, Japan). We used y-GCSh, y-GCSI, MRP, cMOAT, and P-actin oligonucleotides as the probes as described previously [15]. We used topo I oligonucleotides as the probe and the primers were: forward 5'-GAAGTCCGGCATGATAACAA-3' and reverse S'-GTTTGTTAAGACTTGCTGCC-S'. The topo I PCR products were 1208 bp long. In each sample the radioactivity associated with the gene expression level was expressed relative to the p-actin expression level.
Statistical analysis RNA extraction and reverse transcription Total cellular RNA was extracted using the guanidinium isothiocyanate-phenol method and cDNA was synthesized using random hexamer primers (Amersham, Buckinghamshire, UK) with Superscript RNase H - reverse transcriptase (GIBCO-BRL, Bethesda, MD, USA) as described previously [16].
Northern blot hybridization Northern blots of total cellular RNA extracted from the lung cancer cell lines were hybridized with cDNA probes for both subunits of y-GCS as described previously [1]. We used y-GCSh and y-GCS light subunit (y-GCSl) oligonucleotides as the probes as described previously [15].
Contingency table analyses based on x2 statistics were used to determine the significance of associations between categorical variables Differences in expression level between two groups of PMN were analyzed using the Mann-Whitney U-test or the Wilcoxon signedranks test. All of the gene expression levels were skewed toward higher expression levels, and were subjected to logarithmic transformation, so that they more closely approximated a normal distribution, before a parametric test (repeated measures of ANOVA) was performed. The statistical calculations and tests were performed using Stat View J4.ll Software (ABACUS Co., CA, USA) and a Macintosh computer. All of the statistical tests were two-sided, the data are expressed as medians and ranges, and differences with /"-values of less than 0.05 were considered significant.
Results Polymerase chain reaction The reverse-transcribed cDNA from each sample was amplified by the polymerase chain reaction (PCR) using primers based on the y-GCSh. y-GCSl, MRP, cMOAT. Topo I and p-actin (internal control) gene sequences. After pre-denaturation at 94 "C for 5 min, the cDNA was added to 5 ul of the PCR mixture: 1 ul 10x PCR buffer (100 mM Tris-HCl, pH 9.0, 500 mM KC1), I ul 15 mM MgCl2, 2 ul distilled water, 0.2 ul 20 mM dNTPs (Takara, Tokyo, Japan). 0.2 ul 50 uM forward primer, 0.2 ul 50 uM reverse primer, and 0.4 ul (0.2 u) Taq polymerase (Promega. Madison. WI. USA). Amplification was carried out using a thermal cycler (Geneamp PCR System 2400; Perkin Elmer Applied Biosystems Division. Norwalk. CO, USA). Each amplification cycle for the reactions comprised denaturation at 94 ~C for 30 s,
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culture flasks (Becton Dickinson Labware, NJ, USA) at a density of 1 x 106 cells/ml. After a 1-hour preincubation period the cells were incubated with 5 ug/ml of cisplatin (CDDP) for one, four, eight and 24 hours. The CDDP concentration was chosen as described previously [1]. CDDP was obtained from the Bristol Myers Co. (Tokyo, Japan).
22 22 24 24 24 20
First, to investigate the baseline expression levels of the genes encoding the subunits of y-GCS, MRP, cMOAT and Topo I in PMN, we compared the expression levels of 14 normal volunteers with those of 32 lung cancer patients who had not been treated with any chemotherapy before this study. Ten of the normal volunteers were male and four female, they were aged from 24 to 37 years (median of 29 years), and six of them were smokers. The lung cancer patients included 25 males and seven females, with an age range of 41 to 82 years (median of 67 years), and almost all (28 of 32) were smokers (Table 2).
457 Table 2. Patient characteristics."
Table 3. Comparison of baseline gene expression levels in normal volunteers to those in lung cancer patients. b
Pt Age Sex Smoker Histol- TNM PS Therapy no. (years) ogy stage
68 82 79 58 59 62 50 59 68 51 69 73 73 76 67 69 62 41 66 76 67 54 44 68 71 61 62 75 59 72 51 65
M M F F M M M M F M M M M F M M M M M F M M M M M M M M M M F F
AD SQ AD AD AD AD AD AD SQ SQ AD AD SQ AD SM AD SM SM SM AD SM SQ AD AD SQ AD SQ SQ AD AD SQ AD
IV
Ilia IV IV 1Mb
Ilia I IV IHb IV IV I IHb I IHb IV
Ilia IHb IV IV IHb IHb 1Mb IV IV IV IV IV IV IV IV IV
0 2 0 4 2 0 0 2 0 0 3 0 0 0 0 0 0 3 1 2 0 0 0 0 0 0 0 0 0 0 0 0
SM5887 BSC BSC BSC RT
Operation Operation BSC
SM5887 SM5887 BSC
Operation BSC
Operation CDDP + CPT-11 CDDP + CPT-11 CDDP + CPT-11 CDDP+VP CBDCA + VP CBDCA CBDCA + VP CDDP + CPT-11 CDDP + CPT-11 CBDCA CBDCA CBDCA CBDCA CBDCA CBDCA CBDCA CBDCA CBDCA
64 50 55 94 332 341 341 87 101 341 394 331 284 296 459 375 459 371
* Blood samples from patients number 1 — 14 were obtained just before each therapy. b SM5887, newdrugofanthracyclin. c Platinum dose which was given during the study period. Abbreviations: M - male; F - female. + patient who had smoked; - patient who had never smoked; SM - small cell carcinoma; AD - adenocarcinoma; SQ squamous cell carcinoma; PS - performance status; BSC - best supportive care; RT - irradiation; CDDP - cisplatin; CPT-11 - irinotecan hydrochloride; CBDCA - carboplatin; VP - etoposide.
There were no differences in the baseline expression levels between the normal volunteers and the lung cancer patients in any of the genes tested (Table 3, MannWhitney U-test). To examine whether y-GCSh and y-GCSl mRNAs are induced in lung cancer cells in response to platinum drugs we exposed PC-9 cells to CDDP before Northern blot analysis. We found that the y-GCSh mRNA levels had increased about three-fold within 24 hours after exposure to CDDP, whereas the y-GCSl mRNA had not changed (Figure 1). These data were used for determining the sampling time points in the next study. We then monitored the expression level of each gene in PMN from 18 advanced-lung-cancer patients who were treated with a platinum drug only or by platinum drug-based chemotherapy (presented in Table 2, patient numbers 15-32). Fifteen of the 18 patients were male and three female, ranging in age from 41 to 76 years old, with a median of 66 years, and almost all of them (15 of 18) were smokers. CDDP and carboplatin (CBDCA) were administered as a one-hour infusion on day 1. The
Gene expression levels
GCSh GCSI MRP cMOAT Topol
Normal volunteers
Lung cancer patients
0.124(0.054-0.225) 0.135(0.036-0.330) 0.241 (0.040-0.487) 0.260(0.096-1.045) 0.399(0.171-0.638)
0.136(0.011-0.262) 0.177(0.011-0.656) 0.196(0.022-0.710) 0.180(0.038-0.950) 0.388 (0.118-1.225)a
The data was expressed as the median (range). a Median (range) of 28 patients; 4 patients (patient numbers 29-32 in Table 2) were not examined.
PC-9 C 1h 4h 8h 24h /-GCSh
4.1Kb 3.2Kb 4.1Kb
Y-GCSl
1.4Kb 28S Figure 1. Expression of y-GCSh and y-GCSl mRNA in PC-9 cells and in PC-9 cells that had been exposed to CDDP, detected by Northern blot analysis. 28S ribosomal RNAs stained by ethidium bromide were used as an internal control. C - cells not exposed to CDDP; 1 —24 h — cells incubated with 5 ug/ml CDDP for 1, 4, 8 and 24 hours.
CDDP dose was 80 mg/m2 and the CBDCA dose was calculated using Calvert's formula: dose (mg/body) = target AUC x (GFR + 25) [17, 18] and we set the target AUC at 7 mg/ml x min [19]. As shown in Table 4, the y-GCSh expression level increased immediately after exposure to a platinum drug (P < 0.0001, repeated measures of ANOVA), but there was no immediate change in the y-GCSl, MRP, cMOATand Topo I expression levels. To examine long-term effects of the platinum drugs we measured the gene expression levels again, 28 days after completion of platinum drug administration. We were able to obtain PMN from only six of the aforementioned 18 patients for the second measurement. Our results demonstrate that mRNA levels of both y-GCSh and y-GCSl were significantly higher 28 days after platinum drug treatment (both P-values were 0.0464, Wilcoxon signed-ranks test, Table 5), although there were still no differences in the MRP, cMOATand Topo I mRNA levels. These results also suggest that the y-GCSh
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
Platinum dose (mg)c
458 Table 4. Gene expression levels within 24 hours after platinum drug administration. Gene
GCSh GCS1 MRP cMOAT Topo Ia
P-value
Gene expression levels 0 hours
6 hours
24 hours
0.096(0.013-0.211) 0.161 (0.011-0.656) 0.187(0.094-0.710) 0.180(0.038-0.575) 0.450(0.118-1.225)
0.166(0.076-0.752) 0.177(0.055-0.588) 0.218(0.050-1.176) 0.174(0.024-0.543) 0.737(0.309-1.297)
0.238 (0.059-0.533) 0.169(0.028-0.719) 0.189(0.046-2.140) 0.190(0.043-0.567) 0.777(0.203-1.378)
< 0.0001 NS NS NS NS
Abbreviation: NS - not significant. The data was expressed as the median (range). P-values of less than 0.05 were considered to be significant using repeated measures of ANOVA. a Topo I expression levels were calculated from 28 patients; 4 patients (patient numbers 29-32 in Table 2) were not examined.
P-value
Gene expression levels
GCSh GCS1 MRP cMOAT Topo I
Before
After 24 hours
After 28 days
0.140(0.038-0.176) 0.120(0.059-0.245) 0.204(0.101-0.710) 0.220 (0.063-0.575) 0.462(0.331-0.820)
0.343(0.176-0.533) 0.151 (0.044-0.351) 0.384(0.044-1.203) 0.209(0.110-0.422) 0.753 (0.203-0.988)
0.340 (0.074-0.375) 0.301 (0.199-0.915) 0.396(0.161-1.145) 0.211(0.107-0.713) 0.627(0 255-1.111)
0.0464 0.0464 NS NS NS
Abbreviation: NS - not significant. The data from six patients (patient numbers 15, 17, 18, 20-22 in Table 2) was expressed as the median (range). P-values of less than 0.05 were considered to be significant using Wilcoxon signed-ranks test.
gene was induced at 24 hours and that the induction continued for 28 days, whereas the y-GCSl gene showed a delayed response. Discussion In this study we demonstrated that the gene expression levels for both subunits of y-GCS had increased after platinum drug exposure, whereas the MRP, cMOATand Topo I had not. The y-GCSh mRNA level had increased within 24 hours and was still high 28 days after platinum drug exposure. This long-term induction can be explained by the fact that platinum remains in tissues for several months to years after the final exposure [20]. A previous comparison of y-GCSh gene expression in cancer cells and those in their CDDP-resistant cells showed that cells with 1.5- and two-fold increases in y-GCSh expression were 2.9 and 7.2 times more resistant to CDDP, respectively [4]. These results indicate that the 2.5-fold increase in y-GCSh expression that was observed in PMN from lung cancer patients may contribute to platinum drug resistance. Moreover, a transfection study showed that y-GCSh overexpression results in an increased GSH content and platinum drug resistance [5]. When considered together with these reports, our finding of increased y-GCSh mRNA levels after platinum drug exposure suggests a difficulty in anticancer chemotherapy since platinum drug administration itself seems to induce GSH-related platinum drug resistance.
We also found that y-GCSh gene expression increased immediately after CDDP exposure in lung cancer cells in vitro, similar to the results in PMN. Previously O'Dwyer [21] reported that y-GCSh gene expression in the colon correlates with that in PMN. Furthermore, y-GCSh gene expression levels in cancer cells may be associated with resistance to platinum drugs in vitro [22]. These and our present findings indicate that y-GCSh gene expression in PMN can be used as a surrogate marker for resistance, or detoxifying activity, to platinum drugs. Unlike the y-GCSh gene, the y-GCSl gene was not rapidly induced, but the y-GCSl mRNA level had increased 28 days after platinum drug exposure. Since y-GCSh exhibits all of the catalytic activity of the enzyme in GSH synthesis as well as feedback inhibition of GSH [23], rapid consumption of GSH to detoxify the platinum may contribute to the rapid induction of y-GCSh without affecting y-GCSl expression. Whereas, y-GCSl plays a regulatory role, modifying the kinetic properties of y-GCSh [24], increased y-GCSl expression may be needed to keep the y-GCSh expression level high for a long period. The effect of irinotecan hydrochloride (CPT-11) or etoposide (VP-16) on gene induction was a significant problem. Because we had not obtained data from patients who received CPT-11 alone or VP-16 alone, we could not actually describe the effects of these drugs on gene expression. However, our reexamination of 10 patients who were administrated CBDCA alone (patient numbers 20, 24—32 in Table 2) showed that the changes in the levels of expression of each gene were almost
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Table 5. Comparison of gene expression levels between before and 28 days after completion of platinum drug administration.
459
To further elucidate the roles of GSH, GSH-related enzyme and the GS-X pump in platinum drug sensitivity or resistance we are currently examining gene expression levels in tissue samples obtained during surgery from patients with lung cancer.
15.
16.
Acknowledgements This study was supported by Grants-in-Aid from the Ministry of Education, Science and Culture and the Ministry of Health and Welfare of Japan, and a grant from Osaka Cancer Research Foundation. We are grateful to Drs. N. Saijo and K. Nishio (Pharmacology Division, National Cancer Center Research Institute) for providing the coding region of cDNA for the heavy subunit of human liver y-GCS and the PC-9 cells.
17.
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