【摘要】目的：我们先前发现的一个新的氯乙基亚硝脲的类似物2-氯乙基-3-肌氨酸酰胺-1-亚硝基脲(SarCNU)，是一种通过单胺递质的神经元外转运蛋白(EMT)进入细胞内的选择性的细胞毒素。此药已进入I期临床试验。在本研究中，我们检测EMT在23个人类肿瘤细胞系里的表达水平，以证实EMT的表达是否与SarCNU的细胞毒性有关。方法：应用反转录多聚酶链反应(RT-PCR)检测EMT在肿瘤细胞系的表达。同时也用蛋白质印迹技术检测DNA修复蛋白O⁶-甲基鸟嘌呤-DNA甲基转移酶(MGMT)和切除修复交叉互补鼠修复缺乏基因2(ERCC2)。其结果与用硫酸若丹明B(SRB)抗癌药物比色筛选法检测的SarCNU的细胞毒性相关。结果：几乎所有细胞系都呈EMT表达阳性，其中有5个细胞系(MGR-1，MGR-2，T98-G，SK-1，和GBM)呈低水平表达。虽然在SarCNU细胞毒性和EMT表达水平之间没有显著性的线性关系，但多因素回归分析显示SarCNU细胞毒性与EMT加MGMT及ERCC-2表达之间的相关性有显著意义。结论：本研结果示提示EMT和DNA修复因子MGMT、ERCC2是SarCNU抗人肿瘤细胞系活性的重要决定因素。由于大多数人肿瘤细胞系均显示EMT阳性，因此SarCNU可赞助成为治疗人类肿瘤的一种有效的新化疗药物。

中图分类号：R73-34文献标识码：A文章编号：1000-467X(1999)05-0504-05

Extraneuronal monoamine transporter expression in human tumor cell lines

CHEN Zhong-ping

Cancer Center,Sun Yat-sen University of Medical Sciences,Guangzhou 510060, P.R. China

Gerard MohrLawrence C. Panasci

Lady Davis Institute for Medical Research,Sir Mortimer B.Davis-Jewish General Hospital,McGill University,3755 Cte Ste Catherine,Montreal,Quebec,H3T 1E2,Canada

【Abstract】 Objective：We previously have found that 2-chloroethyl-3-sarcosinamide-1-nitrosourea (SarCNU),a new chloroethylnitrosourea analogue, which is in phase I clinical trials,is a selective cytotoxin that enters cells via the extraneuronal transporter for monoamine transmitters (EMT). In the present study,we determined EMT expression in 23 human tumor cell lines in order to verify if EMT expression correlate to SarCNU cytotoxicity. Methods：Reverse-transcription polymerase chain reaction (RT-PCR) was used for determination of EMT expression in tumor cell lines.We also measured expressions of DNA repair protein O⁶-methylguanine-DNA methyltransferase (MGMT) and excision repair cross-complementing rodent repair deficiency gene 2 (ERCC2) by Western blot analysis. The results were correlated to SarCNU cytotoxicity which was determined by the sulforhodamine B (SRB) colorimetric anticancer-drug screening assay.Results：Almost all of the cell lines tested were positive for EMT expression,while five cell lines (MGR-1,MGR-2,T98-G,SKI-1,and GBM) were very low expressors. Although there was no significant linear correlation between SarCNU cytotoxicity and EMT expression,multiple regression analysis demonstrated a significant correlation between SarCNU cytotoxicity and EMT plus MGMT and ERCC-2 expression.Conclusions：This study suggests that both EMT and DNA repair factors, specifically,MGMT and ERCC2 are important determinants of SarCNU activity against human tumor cell lines.SarCNU should prove to be a more useful alternative chemotherapeutic agent for treatment of human tumors,since the majority of human tumor cell lines were EMT positive.

Key words：Extraneuronal monoamine transporter; 2-Chloroethy1-3-sarcosinamide-1-nitrosourea; Human tumor cell line

Extraneuronal transporter for monoamine transmitters (EMT),originally named uptake²,exists in various cells including glia cells of the human central nervous system and some tumor cells^〔1～2〕. We have found that 2-chloroethyl-3-sarcosinamide-1-nitrosourea (SarCNU),a new chloroethylnitrosourea (CENU)analogue^〔3〕,which in phase I clinical trials, is a selective cytotoxin that enters cells via the EMT^〔3〕. Our previous in vitro and in vivo studies demonstrated that SarCNU was more effective than BCNU against human gliomas^〔4～8〕. Using transport studies with radiolabeled SarCNU we previously revealed that SKI-1 cells were negative whereas SKMG-1 cells were positive for the uptake2 transporter,in which SarCNU was more active in SKMG-1 than in SKI-1, suggesting that increased anticancer activity of SarCNU may due to EMT transporter.

Previously,all the results for uptake2 expression were determined biochemically,which is complicated. Recently,human EMT cDNA has been cloned from a human renal cancer cell line Caki-1^〔9〕.In the present investigation, utilizing reverse-transcription polymerase chain reaction (RT-PCR),we have determined EMT expression for 23 human tumor cell lines, and the results have been correlated with cytotoxicity to SarCNU.

1 MATERIALS AND METHODS

1.1 Cell lines

Twenty three established human tumor cell lines were used in this study： SF-295,ACHN,A-498,786-0,Caki-1,SW-620 and SF-767 (National Cancer Institute,USA);T98-G (Dr.D.Yarosh,Applied Genetics Inc., New York,USA);SKMG-1 and SKMG-4 (Dr.G.Cairncross,University of Western Ontario,Canada);SKI-1 (Dr.J.Shapiro,Barrow Neurological Institute,Phoenix,AZ,USA);UW-28,MGR-1,MGR-2 and MGR-3 (Dr.F.Ali-Osma,UT M.D.Anderson Cancer Center,Houston,TX,USA);SKNSH (Dr.E.Shoubridge,Montreal Neulorogical Institute,Canada);SHG-44,GBM and 297 (Dr.Q.Huang,Suzhou Medical College,P.R. China),and HepG2.All cell lines were grown and maintained as cell monolayers in appropriate medium (McCoys 5A supplemented with 10％ fetal bovine serum (FBS),RPMI 1640 supplemented with 5％ FBS, or Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10％ FBS) containing 10 μg/ml gentamycin,in a humidified 5％ CO₂ atmosphere at 37℃ . All cells were harvested for experiments at approaching confluence or confluence state.

1.2 Determination of EMT expression

The method of RT-PCR was used to determine EMT expression in the cell lines.Total RNA was extracted using the RNeasy Midi Kit (Qiagen Inc., Valencia,CA) following manufacturer's protocol. The complementary DNA (cDNA) was synthesized using previous method^〔10〕.Briefly,1 μg total RNA and 50 ng oligo-dT (Pharmacia,Piscataway,NJ) were added in a total volume of 6 μl, heated to 80℃ for 3 minutes,37℃ for 5 minutes, and chilled on ice for 10 minutes.This was followed by the addition of 4 μ l RT-Mix [1 μl 10× PCR buffer,1 μl 2.5 mM dNTPs,16 units Moloney Murine Leukemia Virus Reverse Transcriptase (M-MuLV), and 2 units RNase inhibitor (Pharmacia)] and incubation at 37℃ for 1 hour.Primers were designed using the primer 3 program (Steve Rozen Helen J. Skaletky 1996-1997),and synthesized by Canadian Life Technologies (Burlington,ON).The left primer spun from position 631-650 (5'-3',gcaccaaacttccctgtgtt),and the right primer spun from position 963-944 (5'-3',agcaatgcgtctcaggatct).The reaction volume of 50 μl consists of 2.5 μl of 2.5 mM dNTPs,2 units of DNA polymerase Ampli Tag (pharmacia),20 pmol of each primer,and 2 μl cDNA preparation (synthesised from 0.2 μg total RNA) in 1× PCR buffer (Pharmacia). The PCR cycle comprised 35 cycles of denaturation at 94℃ for 1 minute, annealing at 60℃ for 30 seconds,and elongation at 72℃ for 45 seconds and was run using a PTC-100TM programmable thermal controller (MJ Research Inc., Watertown,MA). Beta actin expression was determined as previously described and was used for normalization^〔10〕. The PCR products were run on 1％ agarose gel and was quantified by using HP ScanJet 5100C Scanner (Hewle