人骨肉瘤U-2 OS细胞甲氨蝶呤（MTX）耐药株的建立及耐药机制的研究
摘要
目的:建立耐甲氨蝶呤(methotrexate,MTX)的人骨肉瘤U-2 OS细胞亚系,探究还原性叶酸载体(reduced folate carrier,RFC)、二氢叶酸还原酶(dihydrofolatereductase,DHFR)基因表达与MTX耐药的关系。
方法:采用大剂量冲击与逐步增加剂量相结合的方法,对人骨肉瘤细胞株U-2 OS进行诱导培养,建立4株耐药细胞株U-2 OS/R1～U-2 OS/R4。MTT法测定各细胞亚系对MTX的敏感性;半定量RT-PCR方法检测各亚系还原性叶酸载体(RFC)、二氢叶酸还原酶(DHFR)mRNA的表达。
结果:本实验成功建立了4株MTX耐药细胞亚系U-2 OS/R1～U-2 OS/R4,MTT显示各亚系的耐药性逐渐增加,与其亲代细胞相比耐药指数RF分别为4、23、71、142倍。随体外U-2 OS/R1～4细胞耐药性增加,RFC基因表达逐渐减少,U-2 OS/R1～R4细胞分别为原代U-2 OS细胞的72%、66%、64%、58%(P均＜0.05);DHFR mRNA表达逐渐增强,DHFR mRNA表达水平在U-2 OS/R1～4细胞分别为原代U-2 OS细胞的3.5、6.2、18.0、31.2倍(p均＜0.05)。
结论:还原性叶酸载体(RFC)、二氢叶酸还原酶(DHFR)mRNA共同参与了U-2 OS骨肉瘤细胞MTX耐药,MTX耐药与RFC基因mRNA表达降低及DHFR基因mRNA表达增加相关。这种现象可能存在于临床的骨肉瘤病人,因而在诊断时测定RFC、DHFR基因状态将有助于筛选MTX耐药病人;而且这些耐药细胞亚系为进一步研究骨肉瘤MTX耐药及逆转提供了一种新的模型。
Objective:To evaluate the impact of reduced folate carrier(RFC) and dihydrofolate reductase(DHFR) genes on methotrexate(MTX) resistance in osteosarcoma cells(U-20S).
Methods:Four MTX-resistant variants(U-2 OS/R1～U-2 OS/R4) were obtained by exposing parental cell line to in vitro high dose and stepwise increased MTX concentrations.The sensitivity of the MTX resistance was measured by MTT assay;the level of reduced folate carrier(RFC) and dihydrofolate reductase(DHFR) mRNA expression was assayed with RT-PCR.
Results:The authors developed four MTX-resistant variants(U-2 OS/R1～U-2 OS/R4) that were resistant to MTX,the resistance factor(RF) in U-2 OS/R1～U-2 OS/R4 is 4,23,71,142 competed with the U-2 OS cell.RFC gene mRNA expression level of resistant cells was decreased gradually along with the concentration increasing of MTX in U-2 OS/R1～U-2 OS/R4 cell lines(72%、66%、64%、58%)(p＜0.05);with DHFR gene mRNA expression level increased 3.5,6.2, 18.0,31.2 folds(p＜0.05).
Conclusions:The MTX resistance was associated with increased levels of DHFR and decrease of RFC gene expression.The DHFR and RFC gene expression level result in MTX resistance in human osteosarcoma cell U-2 OS which can be present at clinical onset in osteosarcoma patients.Simultaneous evaluation of RFC, DHFR gene status at the time of diagnosis may help for the identification of potentially MTX-unresponsive osteosarcoma patients;moreover these MTX resistent osteosarcoma cell sublines are useful models for further research of MTX resistence in osteosarcoma and for the development of treatment protocols.
方法:采用大剂量冲击与逐步增加剂量相结合的方法,对人骨肉瘤细胞株U-2 OS进行诱导培养,建立4株耐药细胞株U-2 OS/R1～U-2 OS/R4。MTT法测定各细胞亚系对MTX的敏感性;半定量RT-PCR方法检测各亚系还原性叶酸载体(RFC)、二氢叶酸还原酶(DHFR)mRNA的表达。
结果:本实验成功建立了4株MTX耐药细胞亚系U-2 OS/R1～U-2 OS/R4,MTT显示各亚系的耐药性逐渐增加,与其亲代细胞相比耐药指数RF分别为4、23、71、142倍。随体外U-2 OS/R1～4细胞耐药性增加,RFC基因表达逐渐减少,U-2 OS/R1～R4细胞分别为原代U-2 OS细胞的72%、66%、64%、58%(P均＜0.05);DHFR mRNA表达逐渐增强,DHFR mRNA表达水平在U-2 OS/R1～4细胞分别为原代U-2 OS细胞的3.5、6.2、18.0、31.2倍(p均＜0.05)。
结论:还原性叶酸载体(RFC)、二氢叶酸还原酶(DHFR)mRNA共同参与了U-2 OS骨肉瘤细胞MTX耐药,MTX耐药与RFC基因mRNA表达降低及DHFR基因mRNA表达增加相关。这种现象可能存在于临床的骨肉瘤病人,因而在诊断时测定RFC、DHFR基因状态将有助于筛选MTX耐药病人;而且这些耐药细胞亚系为进一步研究骨肉瘤MTX耐药及逆转提供了一种新的模型。
Objective:To evaluate the impact of reduced folate carrier(RFC) and dihydrofolate reductase(DHFR) genes on methotrexate(MTX) resistance in osteosarcoma cells(U-20S).
Methods:Four MTX-resistant variants(U-2 OS/R1～U-2 OS/R4) were obtained by exposing parental cell line to in vitro high dose and stepwise increased MTX concentrations.The sensitivity of the MTX resistance was measured by MTT assay;the level of reduced folate carrier(RFC) and dihydrofolate reductase(DHFR) mRNA expression was assayed with RT-PCR.
Results:The authors developed four MTX-resistant variants(U-2 OS/R1～U-2 OS/R4) that were resistant to MTX,the resistance factor(RF) in U-2 OS/R1～U-2 OS/R4 is 4,23,71,142 competed with the U-2 OS cell.RFC gene mRNA expression level of resistant cells was decreased gradually along with the concentration increasing of MTX in U-2 OS/R1～U-2 OS/R4 cell lines(72%、66%、64%、58%)(p＜0.05);with DHFR gene mRNA expression level increased 3.5,6.2, 18.0,31.2 folds(p＜0.05).
Conclusions:The MTX resistance was associated with increased levels of DHFR and decrease of RFC gene expression.The DHFR and RFC gene expression level result in MTX resistance in human osteosarcoma cell U-2 OS which can be present at clinical onset in osteosarcoma patients.Simultaneous evaluation of RFC, DHFR gene status at the time of diagnosis may help for the identification of potentially MTX-unresponsive osteosarcoma patients;moreover these MTX resistent osteosarcoma cell sublines are useful models for further research of MTX resistence in osteosarcoma and for the development of treatment protocols.
引文
1 Davis AM,Bell RS,Goodwin PJ.Prognostic factor in osteosarcoma:a critical review[J].Clin Ontol 1994:12:423-431.
2 Bacci G,Briccoli A,Ferrari Set al.Neoadjuvant chemotherapy for osteosarcoma of the extremities with synchronous lung metastases:treatment with cisplatin,adriamycin and high dose of methotrexate and ifosfamide[J].Oncol Rep 2000;7:339-346.
3 Meyers,PA,Heller q Healey JH et al.Chemotherapy for nonmetastatic osteogenicsarcoma:the Memorial Sloan-Kettering experienceEJ].Clin.Oncol.1992;10:5-15.
4 Matherly LH,Goldman DI.Membrane transport of folates[J]Vitam Horm.2003:66:403-56.
5 Slansky JE,Li Y,Kaelin WG et al.A protein synthesis-dependent increase in E2F1 mRNA correlates with growth regulation of the dihydrofolate reductase promoter[J].Mol Cell Biol 1993;13:1b10-1618.
6 陈静,顾龙君,汤静燕等53例急性白血病患者还原性叶酸载体功能的测定[J]中华血液学杂志2001年02期,100-101
7 陈静,顾龙君,应大明等Namalwa 12/MTX耐药株的建立及其耐药机理的初步研究[J]2002,21(12):1335-1340.
8 张文韬,屠重棋,刘洋,李胜富 人成骨肉瘤顺铂耐药细胞系的建立[J]四川大学学报(医学版),2006,(02),262-265
9 Rosen G,Huvos AG,Mosende C et al.chemotherapy and the rationale for the timing of thoracic surgery[J].Cancer,1978,41(3):841-9.
10 Jaffe N.Recent advances in the chemotherapy of metastatic osteogenic sarcoma [J].Cance 1972,30:1627-1631
11 Delepine N,Delepine G,Jasmin C.et al.Importance of age and methotrexate dosage:Progrnosis in children and young adults with high-grade osteosarcomas [J].Biomed Pharmacother 1988,42:257-262.
12 Huvos AG,Rosen G,Marcove RC.Primary osteogenic sarcoma:Pathologic as pects in 20patients after treatment with enblocresection and prosthetic bone r eplacement[J].Arch PatholLab Med1977,101:14-18.
14 Bacci G,Loro L,Longhi A,et al.No correlation between methotrexate serum level and histologic response in the pre-operative treatment of extremity ost eosarcoma[J].Anticancer33 Drugs.2006,17(4):411-415.
13 Graf N,Winkler K,Betlemovic M.et al.Methotrexate pharmacokinetics and p rognosis in osteosarcoma[J].Clin Oncol,1994.12:1443-1451.
15 Guo W,Healey JH,Meyers PA,et al.Mechanisms of methotrexate resistance in osteosarcoma[J]Clin Cancer Res.1999 Mar;5(3):621-7.
16 邓高荣,李书忠 还原性叶酸载体与骨肉瘤甲氨蝶呤化疗[J]国外医学骨科学分册2005年第03期
17 Hattinger CM,Reverter-Branchat q Remondini D et al.Genomic imbalances associated with methotrexate resistance in human osteosarcoma cell lines detected by comparative genomic hybridization-based techniques[J].Eur J Cell Biol.2003Sep;82(9):483-93.
18 张太娥:陈力军RFC在MTX治疗急性白血病中的作用及研究进展[J]。山东医药2003年11期,049
19 Matherly LH,Taub JW,Ravindranath Y,et al.Elevated dihydrofolate reductase and impaired methot rexate t ransport as elements in methot rexate resistance in childhood acute lymphoblastic leukemia[J].Blood,1995,85(2):50025091
20 Dicker AP,Waltham MC,Volkenands M,et al.Methot rexate resistance in an in vivo mouse tumor due to anon2 active2site dihydrofolate reductase mutation[J].ProcNat 1 Acad Sci USA,1993,90(24):117972-118011
21 Ifergan I,Meller I,Issakov J,Assaraf YG.Reduced folate carrier protein expression in osteosarcoma:implications for the prediction of tumor chemo sensitivity.[J]Cancer.2003 Nov 1;98(9):1958-66.
1 Link MP,GoorinAM,Horowitz Met al.Adjuvant chemotherapy of high-grade osteosarcoma of the extremity:updated results of the Multi-Institutional Osteosarcoma Study[J].Clin Orthop 1991;270:80-14.
2 Saeter qElomaa I,Wahlqvist Y et al.Prognostic factors in bone sarcomas[J].Acta Orthop Scand Suppl 1997;273:156-160.
3 Meyers,PA,Heller q Healey JH et al.Chemotherapy for nonmetastatic oste ogenicsarcoma:the Memorial Sloan-Kettering experience[J].Clin.Oncol.1992;10:5-15.
4 Rosen G,Huvos AG,Mosende C et al.chemotherapy and the rationale for the timing of thoracic surgery[J].Cancer,1978,41(3):841-9.
5 Jaffe N.Recent advances in the chemotherapy of metastatic osteogenic sarcoma [J].Cance 1972,30:1627-1631
6 Delepine N,Delepine G,Jasmin C.et al.Importance of age and methotrexate dosage:Progrnosis in children and young adults with high-grade osteosarcomas[J].Biomed Pharmacother 1988,42:257-262.
7 Graf N,Winkler K,Betlemovic M.et al.Methotrexate pharmacokinetics and prognosis in osteosarcoma[J].Clin Oncol,1994.12:1443-1451.
8 Bacci G,Ferrari S,Delepine N.et al.Predictive factors of histologic response to primary chemotherapy in osteosarcoma of the extremity: Study of 272 patients preoperatively treated with high dose methotrexate, doxorubicin and cisplatin [J].Clin Oncol, 1998,16(2):658-663.
9 Banerjee D,Mayer-Kuckuh P, Capiaux G et al.Novel aspects of resistance to drugs targeted to dihydrofolate reductase and thymidylate synthase. [J].Biochim Biophys Acta 2002;1587:154-173.
10 Sowers R, Toguchida J, Qin J, Meyers PA et al. mRNA expression levels of E2 F transcription factors correlate with dihydrofolate reductase, reduced folate carrier, andthymidylate synthase mRNA expression in osteosarcoma. [J] Mol Cance r Ther 2003, Jun; 2[6]:535-41
11 Moscow JA. Methotrexate transport and resistance[J]. Leuk Lymphoma. 1998 Jul; 30(3-4):215-24.
12 Sirotnak FM, Tolner B. Carrier-mediated membrane transport of folates in mammalian cells.Annu Rev Nutr. 1999;19:91-122.
13 Matherly LH, Goldman DL. Membrane transport of folates[J]. Vitam Horm. 2003,66:403-56
14 Brzezinska A. Winska P, Balinska M . Cellular aspects of folate and antifolate membrane transport[J]. Acta Biochim Pol.2000:47(3):735-49.
15 Home DW Transport of folates and antifolates in liver[J]. Proc Soc Exp Biol Med.1993 Apr;202(4):385-91,
16 Slansky JE, Li Y, Kaelin WG et al.A protein synthesis-dependent increase in E2F1 mRNA correlates with growth regulation of the dihydrofolate reductase promoter[J].Mol Cell Biol 1993;13:1610-1618.
17 Prasad PD, Ramamoorthy S, Leibach FH, Ganapathy V Molecular cloning of the human placental folate transporter[J]. Biochem Biophys Res Commun.1995 Jan 17;206(2):681-7.
18 WhetstineJR, MatherlyLH. The basal promoters for the human re-duced folate carrier gene are regulated bya GC-box and a cAMP-response element, Basis for tissue-specific gene expression[J]. Bio, 2001;276(9):6350-6358.
19 Zhao R, Seither R, Brigle KE et al. Impact of overexpression of the reduced folate carrier (RFC 1), an anion exchanger, on concentrative transport in murine L 1210 leukemiacells[J].Biol Chem. 1997 Aug 22;272(34):21207-12.
20 Moscow JA, Gong M, He R, Sgagias MK et al. Isolation of a gene encoding a human reduced folate carrier (RFC 1)and analysis of its expression in transport-def cient, methotrexate-resistant human breast cancer cells[J]. Cancer Res.1995 Sep 1;55(17):3790-4.
21 Kaufman Y Drori S, Cole PD et al. Reduced fohte carrier mutations are no t the mechanism underlying methotrexate resistance in childhood acute lymphobl astic leukemia. [J].Cancer 2004 Feb 15; 100(4):773-82
22 Ding BC,WhetstineJR, Witt TL, Schuetz JD,Matherly LH. Repression of human reduced folate carrier gene expression by wild type p53. [J]. Biol Chem. 2001 Mar 23;276(12):8713-9.
23 Jesper Worm, Alexei F. Kirkin, Karine N. Dzhandzhugazyan, and PerGuldberg Methylation-dependent Silencing of the Reduced Folate Carrier Gene inlnherently Methotrexate-resistant Human Breast Cancer Cells. [J] Bio Chem. 2001 Oct 26;276(43):39990-40000.
24 Wei Guo,John H. Healey, Paul A.Meyers et al. Mechanisms of Methotrexate Resistance in Osteosarcoma[J]. Clinical,1999 March;Vol. 5, 621-627.
25 Hattinger CM,Reverter-Branchat q Remondini D et al.Genomic imbalances associated with methotrexate resistance in human osteosarcoma cell lines detected by comparative genomic hybridization-based techniques[J]. Eur J Cell Biol. 2003 Sep;82(9):483-93.
26 Ilan Ifergan, Issac Meller, Josefin Issakow et al. Reduced folate carrier protein expression in osteosarcoma implications for the prediction of tumor chemosensitivity[J].Cancer. 2003 Nov 1; 98(9):1958-66.
27 Serra M, Reverter-BranchatqMaurici D. Analysis of dihydrofolate reductase and reduced folate carrier gene status in relation to methotrexate resistance in osteosarcoma cells[J]. Ann Oncol. 2004 Jan; 15(1):151-60.
28 Rui Yang, Rebecca Sowers, BethAnne Mazza et al. Sequence Alterations in the Reduced Folate Carrier Are Observed in Osteosarcoma Tumor Samples[J]. Clinical Cancer Research. 2003 February; Vol.9, 837-844.
29 Waltham M, Kheradpour A, et al. Amplification of the dihydrofolate reductase gene is mechanism of acquired resistance to methotrexate in patient with acute lymphoblastic leukemia and is correlated with p53 gene mutations[J]. Blood, 1995,86:6772684.
30 Diddens H, N iethammer D, Jackson RC.Patterns of cross-resistance tothe antifolate drugs trimetrexate, metoprine, homofolate, and CB3717 in human lymphoma and osteosarcoma cells resistant to methotrexate[J]. Cancer Res. 1983 Nov;43(11):5286-92.
31 Levy AS, Sather HN, Steinherz PG, et al. Reduced folate carrier and dihydrofolate reductase expression in acute lymphocytic leukemia may predict outcome:a Children' s Cancer Group Study[J].Pediator Hematol Oncol,2003,25(9):6882695.
32 陈静 顾龙君,汤静燕等53例急性白血病患者还原性叶酸载体功能的测定[J],中华血液学杂志2001年02期,100-101
33 陈静 顾龙君,应大明等Namalwa 12/MTX耐药株的建立及其耐药机理的初步研究[J].2002,21(12):1335-1340.
2 Bacci G,Briccoli A,Ferrari Set al.Neoadjuvant chemotherapy for osteosarcoma of the extremities with synchronous lung metastases:treatment with cisplatin,adriamycin and high dose of methotrexate and ifosfamide[J].Oncol Rep 2000;7:339-346.
3 Meyers,PA,Heller q Healey JH et al.Chemotherapy for nonmetastatic osteogenicsarcoma:the Memorial Sloan-Kettering experienceEJ].Clin.Oncol.1992;10:5-15.
4 Matherly LH,Goldman DI.Membrane transport of folates[J]Vitam Horm.2003:66:403-56.
5 Slansky JE,Li Y,Kaelin WG et al.A protein synthesis-dependent increase in E2F1 mRNA correlates with growth regulation of the dihydrofolate reductase promoter[J].Mol Cell Biol 1993;13:1b10-1618.
6 陈静,顾龙君,汤静燕等53例急性白血病患者还原性叶酸载体功能的测定[J]中华血液学杂志2001年02期,100-101
7 陈静,顾龙君,应大明等Namalwa 12/MTX耐药株的建立及其耐药机理的初步研究[J]2002,21(12):1335-1340.
8 张文韬,屠重棋,刘洋,李胜富 人成骨肉瘤顺铂耐药细胞系的建立[J]四川大学学报(医学版),2006,(02),262-265
9 Rosen G,Huvos AG,Mosende C et al.chemotherapy and the rationale for the timing of thoracic surgery[J].Cancer,1978,41(3):841-9.
10 Jaffe N.Recent advances in the chemotherapy of metastatic osteogenic sarcoma [J].Cance 1972,30:1627-1631
11 Delepine N,Delepine G,Jasmin C.et al.Importance of age and methotrexate dosage:Progrnosis in children and young adults with high-grade osteosarcomas [J].Biomed Pharmacother 1988,42:257-262.
12 Huvos AG,Rosen G,Marcove RC.Primary osteogenic sarcoma:Pathologic as pects in 20patients after treatment with enblocresection and prosthetic bone r eplacement[J].Arch PatholLab Med1977,101:14-18.
14 Bacci G,Loro L,Longhi A,et al.No correlation between methotrexate serum level and histologic response in the pre-operative treatment of extremity ost eosarcoma[J].Anticancer33 Drugs.2006,17(4):411-415.
13 Graf N,Winkler K,Betlemovic M.et al.Methotrexate pharmacokinetics and p rognosis in osteosarcoma[J].Clin Oncol,1994.12:1443-1451.
15 Guo W,Healey JH,Meyers PA,et al.Mechanisms of methotrexate resistance in osteosarcoma[J]Clin Cancer Res.1999 Mar;5(3):621-7.
16 邓高荣,李书忠 还原性叶酸载体与骨肉瘤甲氨蝶呤化疗[J]国外医学骨科学分册2005年第03期
17 Hattinger CM,Reverter-Branchat q Remondini D et al.Genomic imbalances associated with methotrexate resistance in human osteosarcoma cell lines detected by comparative genomic hybridization-based techniques[J].Eur J Cell Biol.2003Sep;82(9):483-93.
18 张太娥:陈力军RFC在MTX治疗急性白血病中的作用及研究进展[J]。山东医药2003年11期,049
19 Matherly LH,Taub JW,Ravindranath Y,et al.Elevated dihydrofolate reductase and impaired methot rexate t ransport as elements in methot rexate resistance in childhood acute lymphoblastic leukemia[J].Blood,1995,85(2):50025091
20 Dicker AP,Waltham MC,Volkenands M,et al.Methot rexate resistance in an in vivo mouse tumor due to anon2 active2site dihydrofolate reductase mutation[J].ProcNat 1 Acad Sci USA,1993,90(24):117972-118011
21 Ifergan I,Meller I,Issakov J,Assaraf YG.Reduced folate carrier protein expression in osteosarcoma:implications for the prediction of tumor chemo sensitivity.[J]Cancer.2003 Nov 1;98(9):1958-66.
1 Link MP,GoorinAM,Horowitz Met al.Adjuvant chemotherapy of high-grade osteosarcoma of the extremity:updated results of the Multi-Institutional Osteosarcoma Study[J].Clin Orthop 1991;270:80-14.
2 Saeter qElomaa I,Wahlqvist Y et al.Prognostic factors in bone sarcomas[J].Acta Orthop Scand Suppl 1997;273:156-160.
3 Meyers,PA,Heller q Healey JH et al.Chemotherapy for nonmetastatic oste ogenicsarcoma:the Memorial Sloan-Kettering experience[J].Clin.Oncol.1992;10:5-15.
4 Rosen G,Huvos AG,Mosende C et al.chemotherapy and the rationale for the timing of thoracic surgery[J].Cancer,1978,41(3):841-9.
5 Jaffe N.Recent advances in the chemotherapy of metastatic osteogenic sarcoma [J].Cance 1972,30:1627-1631
6 Delepine N,Delepine G,Jasmin C.et al.Importance of age and methotrexate dosage:Progrnosis in children and young adults with high-grade osteosarcomas[J].Biomed Pharmacother 1988,42:257-262.
7 Graf N,Winkler K,Betlemovic M.et al.Methotrexate pharmacokinetics and prognosis in osteosarcoma[J].Clin Oncol,1994.12:1443-1451.
8 Bacci G,Ferrari S,Delepine N.et al.Predictive factors of histologic response to primary chemotherapy in osteosarcoma of the extremity: Study of 272 patients preoperatively treated with high dose methotrexate, doxorubicin and cisplatin [J].Clin Oncol, 1998,16(2):658-663.
9 Banerjee D,Mayer-Kuckuh P, Capiaux G et al.Novel aspects of resistance to drugs targeted to dihydrofolate reductase and thymidylate synthase. [J].Biochim Biophys Acta 2002;1587:154-173.
10 Sowers R, Toguchida J, Qin J, Meyers PA et al. mRNA expression levels of E2 F transcription factors correlate with dihydrofolate reductase, reduced folate carrier, andthymidylate synthase mRNA expression in osteosarcoma. [J] Mol Cance r Ther 2003, Jun; 2[6]:535-41
11 Moscow JA. Methotrexate transport and resistance[J]. Leuk Lymphoma. 1998 Jul; 30(3-4):215-24.
12 Sirotnak FM, Tolner B. Carrier-mediated membrane transport of folates in mammalian cells.Annu Rev Nutr. 1999;19:91-122.
13 Matherly LH, Goldman DL. Membrane transport of folates[J]. Vitam Horm. 2003,66:403-56
14 Brzezinska A. Winska P, Balinska M . Cellular aspects of folate and antifolate membrane transport[J]. Acta Biochim Pol.2000:47(3):735-49.
15 Home DW Transport of folates and antifolates in liver[J]. Proc Soc Exp Biol Med.1993 Apr;202(4):385-91,
16 Slansky JE, Li Y, Kaelin WG et al.A protein synthesis-dependent increase in E2F1 mRNA correlates with growth regulation of the dihydrofolate reductase promoter[J].Mol Cell Biol 1993;13:1610-1618.
17 Prasad PD, Ramamoorthy S, Leibach FH, Ganapathy V Molecular cloning of the human placental folate transporter[J]. Biochem Biophys Res Commun.1995 Jan 17;206(2):681-7.
18 WhetstineJR, MatherlyLH. The basal promoters for the human re-duced folate carrier gene are regulated bya GC-box and a cAMP-response element, Basis for tissue-specific gene expression[J]. Bio, 2001;276(9):6350-6358.
19 Zhao R, Seither R, Brigle KE et al. Impact of overexpression of the reduced folate carrier (RFC 1), an anion exchanger, on concentrative transport in murine L 1210 leukemiacells[J].Biol Chem. 1997 Aug 22;272(34):21207-12.
20 Moscow JA, Gong M, He R, Sgagias MK et al. Isolation of a gene encoding a human reduced folate carrier (RFC 1)and analysis of its expression in transport-def cient, methotrexate-resistant human breast cancer cells[J]. Cancer Res.1995 Sep 1;55(17):3790-4.
21 Kaufman Y Drori S, Cole PD et al. Reduced fohte carrier mutations are no t the mechanism underlying methotrexate resistance in childhood acute lymphobl astic leukemia. [J].Cancer 2004 Feb 15; 100(4):773-82
22 Ding BC,WhetstineJR, Witt TL, Schuetz JD,Matherly LH. Repression of human reduced folate carrier gene expression by wild type p53. [J]. Biol Chem. 2001 Mar 23;276(12):8713-9.
23 Jesper Worm, Alexei F. Kirkin, Karine N. Dzhandzhugazyan, and PerGuldberg Methylation-dependent Silencing of the Reduced Folate Carrier Gene inlnherently Methotrexate-resistant Human Breast Cancer Cells. [J] Bio Chem. 2001 Oct 26;276(43):39990-40000.
24 Wei Guo,John H. Healey, Paul A.Meyers et al. Mechanisms of Methotrexate Resistance in Osteosarcoma[J]. Clinical,1999 March;Vol. 5, 621-627.
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