叶酰聚谷氨酸合成酶在氨甲蝶呤对映体耐药A549细胞株中的表达及临床应用
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摘要
目的:探讨叶酰聚谷氨酸合成酶(FPGS)在氨甲蝶呤(MTX)对映体(L-(+)-MTX和D-(-)–MTX)耐药A549细胞株中的表达,并应用于临床病例。
方法:用SYBR Green I为荧光染料,以β-actin作参照,建立检测FPGS mRNA的实时荧光定量PCR方法;根据循环阈值(Ct值)、标准曲线相关系数、斜率、重复性曲线、熔解曲线、扩增效率曲线等进行方法学评价;并用该方法检测MTX对映体耐药细胞株及应用MTX耐药的白血病骨髓细胞中FPGS基因表达;用蛋白免疫印迹方法测定肺癌A549细胞株和MTX对映体耐药A549细胞株中的FPGS蛋白表达。
结果:建立的标准曲线Ct值与模板浓度有良好的线性关系,FPGS和β-actin标准曲线相关系数分别为0.9968和0.9987,斜率分别为-3.595和-3.740,批内变异系数(CV)为1.27%～2.95%,批间CV为3.82%,熔解曲线均呈单个特异峰,扩增效率相似(斜率为0.0217);15μmol/L、35μmol/L、55μmol/L各浓度点L-(+)-MTX和D-(-)–MTX两组间FPGS mRNA表达水平差异均有统计学意义(0.80±0.09 vs 2.04±0.34,3.43±0.44 vs 8.11±0.87,2.00±0.22 vs 0.59±0.11, P<0.05);15μmol/L L-(+)-MTX和D-(-)–MTX两组间FPGS蛋白表达水平差异有统计学意义(0.85±0.12 vs 1.62±0.24,P<0.05);浓度为35μmol/L、55μmol/L时, L-(+)-MTX和D-(-)–MTX两组间蛋白表达水平差异均无统计学意义(P>0.05);白血病患者应用MTX耐药后FPGS相对含量是用前的0.35±0.04倍,两组间差异具有统计学意义(P<0.05)。
结论:建立的实时荧光定量PCR检测FPGS mRNA的方法重复性好、特异度高、稳定性好、简单易行且成本低廉,可用于FPGS基因含量分析;白血病患者应用MTX耐药前后骨髓细胞中FPGS基因表达发生了变化;MTX诱导耐药后细胞株中FPGS在mRNA和蛋白水平上均发生了变化且在两种对映体细胞株间具有手性差异。
Objective:To study the expression of folylpolyglutamate synthetase (FPGS) in methotrexate enantiomers resistant A549 cell lines (L-(+)-MTX和D-(-)–MTX)and the application of clinical specimens.
Methods:A real-time fluorescence quantitative PCR method for detection of FPGS mRNA was established using SYBR Green I as fluorescence andβ-actin as reference. The method was evaluated by cycle threshold (Ct), correlation coefficient, slope, repeatability curve, melting curve and amplification efficiency curve. The expression levels of FPGS gene in methotrexate enantiomer-resistant A549 cell lines and methotrexate resistant leukemia cells in bone marrow were detected by the method. The expression of FPGS on protein level was determined by Western-Blot in lung cancer A549 cells, and methotrexate enantiomers resistant A549 cells.
Results:The standard curves indicated the high linear relationship between cycle threshold and template concentration was observed. The correlation coefficients of FPGS andβ-actin were 0.9968 and 0.9987, and the slopes were -3.595 and -3.740, respectively. The inter-coefficient of variation was from 1.27% to 2.95%. The intra-coefficient of variation was 3.82%. The method was characterized of specific melting curve and similar amplification efficiency (slope =0.0217). The expression level of FPGS mRNA was statistical different in various MTX concentration resistant cell lines (0.80±0.09 vs 2.04±0.34,3.43±0.44 vs 8.11±0.87,2.00±0.22 vs 0.59±0.11, P<0.05)。The protein expression level of FPGS was 0.85±0.12 and 1.62±0.24 folds in L-(+)-MTX/A549 cells and D-(-)-MTX/A549 cells with the concentration of drug resistance is 15μmol/L ,compared with lung cancer A549 cells.Statistical difference was observed between two groups (P<0.05). However there had no statistical difference in MTX enantiomers resistant cell line with the concentration of drug resistance are 35μmol/L and 55μmol/L (P>0.05). After treated with methotrexate(group B), the expression level of FPGS mRNA was 0.35±0.04 folds compared with those before treatment (group A) among methotrexate resistant leukemia patients. Statistical difference was observed between group A and group B(P<0.05).
Conclusion:The real-time fluorescence quantitative PCR has many advantages, such as a reproductive, specific, stable, simple and cheap method, which is suitable for the quantification of FPGS. The expression levels of FPGS in methotrexate resistant leukemia cells in bone marrow have been changed. The expression level of FPGS in drug resistant cells have been changed, especially significant difference in enantiomer-resistant cells are appeared.
方法:用SYBR Green I为荧光染料,以β-actin作参照,建立检测FPGS mRNA的实时荧光定量PCR方法;根据循环阈值(Ct值)、标准曲线相关系数、斜率、重复性曲线、熔解曲线、扩增效率曲线等进行方法学评价;并用该方法检测MTX对映体耐药细胞株及应用MTX耐药的白血病骨髓细胞中FPGS基因表达;用蛋白免疫印迹方法测定肺癌A549细胞株和MTX对映体耐药A549细胞株中的FPGS蛋白表达。
结果:建立的标准曲线Ct值与模板浓度有良好的线性关系,FPGS和β-actin标准曲线相关系数分别为0.9968和0.9987,斜率分别为-3.595和-3.740,批内变异系数(CV)为1.27%～2.95%,批间CV为3.82%,熔解曲线均呈单个特异峰,扩增效率相似(斜率为0.0217);15μmol/L、35μmol/L、55μmol/L各浓度点L-(+)-MTX和D-(-)–MTX两组间FPGS mRNA表达水平差异均有统计学意义(0.80±0.09 vs 2.04±0.34,3.43±0.44 vs 8.11±0.87,2.00±0.22 vs 0.59±0.11, P<0.05);15μmol/L L-(+)-MTX和D-(-)–MTX两组间FPGS蛋白表达水平差异有统计学意义(0.85±0.12 vs 1.62±0.24,P<0.05);浓度为35μmol/L、55μmol/L时, L-(+)-MTX和D-(-)–MTX两组间蛋白表达水平差异均无统计学意义(P>0.05);白血病患者应用MTX耐药后FPGS相对含量是用前的0.35±0.04倍,两组间差异具有统计学意义(P<0.05)。
结论:建立的实时荧光定量PCR检测FPGS mRNA的方法重复性好、特异度高、稳定性好、简单易行且成本低廉,可用于FPGS基因含量分析;白血病患者应用MTX耐药前后骨髓细胞中FPGS基因表达发生了变化;MTX诱导耐药后细胞株中FPGS在mRNA和蛋白水平上均发生了变化且在两种对映体细胞株间具有手性差异。
Objective:To study the expression of folylpolyglutamate synthetase (FPGS) in methotrexate enantiomers resistant A549 cell lines (L-(+)-MTX和D-(-)–MTX)and the application of clinical specimens.
Methods:A real-time fluorescence quantitative PCR method for detection of FPGS mRNA was established using SYBR Green I as fluorescence andβ-actin as reference. The method was evaluated by cycle threshold (Ct), correlation coefficient, slope, repeatability curve, melting curve and amplification efficiency curve. The expression levels of FPGS gene in methotrexate enantiomer-resistant A549 cell lines and methotrexate resistant leukemia cells in bone marrow were detected by the method. The expression of FPGS on protein level was determined by Western-Blot in lung cancer A549 cells, and methotrexate enantiomers resistant A549 cells.
Results:The standard curves indicated the high linear relationship between cycle threshold and template concentration was observed. The correlation coefficients of FPGS andβ-actin were 0.9968 and 0.9987, and the slopes were -3.595 and -3.740, respectively. The inter-coefficient of variation was from 1.27% to 2.95%. The intra-coefficient of variation was 3.82%. The method was characterized of specific melting curve and similar amplification efficiency (slope =0.0217). The expression level of FPGS mRNA was statistical different in various MTX concentration resistant cell lines (0.80±0.09 vs 2.04±0.34,3.43±0.44 vs 8.11±0.87,2.00±0.22 vs 0.59±0.11, P<0.05)。The protein expression level of FPGS was 0.85±0.12 and 1.62±0.24 folds in L-(+)-MTX/A549 cells and D-(-)-MTX/A549 cells with the concentration of drug resistance is 15μmol/L ,compared with lung cancer A549 cells.Statistical difference was observed between two groups (P<0.05). However there had no statistical difference in MTX enantiomers resistant cell line with the concentration of drug resistance are 35μmol/L and 55μmol/L (P>0.05). After treated with methotrexate(group B), the expression level of FPGS mRNA was 0.35±0.04 folds compared with those before treatment (group A) among methotrexate resistant leukemia patients. Statistical difference was observed between group A and group B(P<0.05).
Conclusion:The real-time fluorescence quantitative PCR has many advantages, such as a reproductive, specific, stable, simple and cheap method, which is suitable for the quantification of FPGS. The expression levels of FPGS in methotrexate resistant leukemia cells in bone marrow have been changed. The expression level of FPGS in drug resistant cells have been changed, especially significant difference in enantiomer-resistant cells are appeared.
引文
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3.Evans WE, Relling MV, Rodman JH, et al. Conventional compared with individualized chemotherapy for childhood acute lymphoblastic leukemia. N Engl J Med. 1998; 338(8):499-505.
4.Ackland SP, Schilsky RL.. High-dose methotrexate: a critical reappraisal[J]. J Clin Oncol. 1987; 5(12):2017–31.
5.Evans WE, Crom WR, Abromowitch M, et al. Clinical pharmacodynamics of high-dose methotrexate in acute lymphoblastic leukemia: identification of a relation between concentration and effect[J]. N Engl J Med. 1986; 314(8):471-7.
6.Niemeyer CM, Gelber RD, Tarbell NJ, et al. Low-dose versus high-dose methotrexate during remission induction in childhood acute lymphoblastic leukemia (Protocol 81-01 update) [J]. Blood. 1991; 78(10):2514-9.
7. Gangjee A ,Dubash NP,Zeng Y et al. Recent Advances in the Chemistry and Biology of Folypoly-g-glutamate Synthetase Substrates and InhibitorsCurr. Med. Chem.–Anti-Cancer Agents, 2002, 2(3):331-332
8.Tomsho JW,Mcguire JJ,Coward JK et al.Synthesis of (6R)- and (6S)-5,10-dideazatetrahydrofolate oligo-γ-glutamates: Kinetics of multiple glutamate ligations catalyzed by folylpoly-γ-glutamate synthetase Org Biomol Chem, 2005, 3(18): 388–3398
9.陈静,顾龙君,应大明,等.急性白血病氨甲蝶呤耐药及化疗个体化初探[J].中华血液学杂志.. 2002; 23(1): 35-6.
10.Sohn KJ , Smirnakis F,Moskovitz DN et al . Effects of folyl-polyglutamate synthetasemodulation on chemosensitivity of colon cancer cells to 5-fluorouracil and met hot rexate [ J ] . Gut , 2004 ,53 (12) : 1825-1831
11.Cramer SM,Schornagel JH,Kalghatgi KK, et al. Occurrence and significance of D-methotrexate as a contaminant of commercial methotrexate[J]. Cancer Res, 1984, 44(5):1843-1846
12.金鑫,沈佐君,何晓东,等.亲和毛细管电泳法测定甲氨蝶呤对映体与二氢叶酸还原酶反应动力学参数[J].中华检验医学杂志,2008,31(3):317-321
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15.Stark M, Wichman C, Avivi I et al. Aberrant splicing of folylpolyglutamate synthetase as a novel mechanism of antifolate resistance in leukemia. Blood, 2009, 113: 4362-4369
16.Aldea C, Alvarev CP, Folgueira L, et al. Rapid detection of herpes simp lex virus in genital ulcers by real2time PCR using SYBR Green dye as the detection signal[ J ]. J ClinMicrobiol, 2002, 40: 1060 -1062.
17.陶绍能,沈佐君,何晓东,等.氨甲喋呤对映体耐药A549细胞株的建立及其生物学特征[J]肿瘤防治研究, 2009,36(4):273-277
18.凡任芝,沈佐君,何晓东,等.毛细管电泳免疫-激光诱导荧光检测技术测定氨甲蝶呤对映体耐药A549细胞株中叶酰聚谷氨酸合成酶.中华检验医学杂志,2008,31(8):432-440
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1. Nair J,Mcguire J. Submitochondrial localization of the mitochondrial isoform of folylpolyglutamate synthetase in CCRF-CEM human T-lymphoblastic leukemia cells Biochim Biophys Acta, 2005, 1746 (1):38-44
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4. Tomsho J,Mcguire J,Coward J. Synthesis of (6R)- and (6S)-5,10-dideazatetrahydrofolate oligo-γ-glutamates: Kinetics of multiple glutamate ligations catalyzed by folylpoly-γ-glutamate synthetase Org Biomol Chem, 2005, 3(18): 388–3398
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8. Izbicka EA,Streeper D,Wick MD et al. Distinct mechanistic activity proWle of pralatrexate in comparison to other antifolates in in vitro and in vivo models of human cancers Cancer Chemother Pharmacol, 2009,64:993–999
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11. Leclerc G,York T,Kinser T et al. Molecular basis for decreased folylpoly-_-glutamate synthetase expression in a methotrexate resistant CCRF-CEM mutant cell line Leuk Res, 2007, 31(3):293-299
12. Wettergren Y,Odin E,Nilsson S et al. Low expression of reduced folate carrier-1 and folylpolyglutamate synthase correlates with lack of a deleted in colorectal carcinoma mRNA splice variant in normal-appearing mucosa of colorectal carcinoma patients Cancer Detection and Prevention, 2005, 29:348–355
13. Stark M ,Wichman C,Avivi I et al. Aberrant splicing of folylpolyglutamate synthetase as a novel mechanism of antifolate resistance in leukemia blood , 2009, 113: 4362-4369
14. Dotzlaf J,Carpenter J,Luo S et al. Derivation and Characterization of Monoclonal Antibodies Against Human Folypolyglutamate Synthetase Hybridoma, 2007, 3(26):155-161
15. Wettergren Y ,Odin E,Nilsson S et al. p16INK4a Gene Promoter Hypermethylation in Mucosa as a Prognostic Factor for Patients with Colorectal Cancer Cancer Research Article, 2008, 14(7-8):412-421
16. Assaraf Y. Molecular basis of antifolate resistance Cancer Metastasis Rev, 2007,26 (1):153-181
17. Gangjee A,Jain H,Kurup S. Recent Advances in Classical and Non-Classical Antifolates as Antitumor and
18. Antiopportunistic Infection Agents: Part IIAnti-Cancer Agents in Medicinal Chemistry, 2008, 8:205-231
2.Schilsky RL. Methotrexate: an effective agent for treating cancer and building careers. The polyglutamate era[J]. Stem Cells. 1996; 14(1): 29-32.
3.Evans WE, Relling MV, Rodman JH, et al. Conventional compared with individualized chemotherapy for childhood acute lymphoblastic leukemia. N Engl J Med. 1998; 338(8):499-505.
4.Ackland SP, Schilsky RL.. High-dose methotrexate: a critical reappraisal[J]. J Clin Oncol. 1987; 5(12):2017–31.
5.Evans WE, Crom WR, Abromowitch M, et al. Clinical pharmacodynamics of high-dose methotrexate in acute lymphoblastic leukemia: identification of a relation between concentration and effect[J]. N Engl J Med. 1986; 314(8):471-7.
6.Niemeyer CM, Gelber RD, Tarbell NJ, et al. Low-dose versus high-dose methotrexate during remission induction in childhood acute lymphoblastic leukemia (Protocol 81-01 update) [J]. Blood. 1991; 78(10):2514-9.
7. Gangjee A ,Dubash NP,Zeng Y et al. Recent Advances in the Chemistry and Biology of Folypoly-g-glutamate Synthetase Substrates and InhibitorsCurr. Med. Chem.–Anti-Cancer Agents, 2002, 2(3):331-332
8.Tomsho JW,Mcguire JJ,Coward JK et al.Synthesis of (6R)- and (6S)-5,10-dideazatetrahydrofolate oligo-γ-glutamates: Kinetics of multiple glutamate ligations catalyzed by folylpoly-γ-glutamate synthetase Org Biomol Chem, 2005, 3(18): 388–3398
9.陈静,顾龙君,应大明,等.急性白血病氨甲蝶呤耐药及化疗个体化初探[J].中华血液学杂志.. 2002; 23(1): 35-6.
10.Sohn KJ , Smirnakis F,Moskovitz DN et al . Effects of folyl-polyglutamate synthetasemodulation on chemosensitivity of colon cancer cells to 5-fluorouracil and met hot rexate [ J ] . Gut , 2004 ,53 (12) : 1825-1831
11.Cramer SM,Schornagel JH,Kalghatgi KK, et al. Occurrence and significance of D-methotrexate as a contaminant of commercial methotrexate[J]. Cancer Res, 1984, 44(5):1843-1846
12.金鑫,沈佐君,何晓东,等.亲和毛细管电泳法测定甲氨蝶呤对映体与二氢叶酸还原酶反应动力学参数[J].中华检验医学杂志,2008,31(3):317-321
13.Wettergren Y, Odin E, Nilsson S et al. Low expression of reduced folate carrier-1 and folylpolyglutamate synthase correlates with lack of a deleted in colorectal carcinoma mRNA splice variant in normal-appearing mucosa of colorectal carcinoma patients. Cancer Detection and Prevention,2005, 29:348–355
14.Leclerc G, York T, Hsieh-Kinser T et al. Molecular basis for decreased folylpolyglutamate synthetase expression in a methotrexate resistant CCRF-CEM mutant cell line .Leuk Res,2007, 31(3):293-299
15.Stark M, Wichman C, Avivi I et al. Aberrant splicing of folylpolyglutamate synthetase as a novel mechanism of antifolate resistance in leukemia. Blood, 2009, 113: 4362-4369
16.Aldea C, Alvarev CP, Folgueira L, et al. Rapid detection of herpes simp lex virus in genital ulcers by real2time PCR using SYBR Green dye as the detection signal[ J ]. J ClinMicrobiol, 2002, 40: 1060 -1062.
17.陶绍能,沈佐君,何晓东,等.氨甲喋呤对映体耐药A549细胞株的建立及其生物学特征[J]肿瘤防治研究, 2009,36(4):273-277
18.凡任芝,沈佐君,何晓东,等.毛细管电泳免疫-激光诱导荧光检测技术测定氨甲蝶呤对映体耐药A549细胞株中叶酰聚谷氨酸合成酶.中华检验医学杂志,2008,31(8):432-440
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