神经元外单胺转运蛋白在人肿瘤细胞系的表达
作者：陈忠平 GerardMohr LawrenceC.Panasci
单位：中山医科大学肿瘤防治中心脑外科(广州，510060)
关键词：神经元外单胺转运蛋白；2-氯乙基-3-肌氨酸胺-1-亚硝基脲；人肿瘤细胞系
癌症990505
【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。
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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 Mohr Lawrence C. Panasci
Lady Davis Institute for Medical Research,Sir Mortimer B.Davis-Jewish General Hospital,McGill University,3755 C
te Ste Catherine,Montreal,Quebec,H3T 1E2,Canada
, 百拇医药
【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.
, 百拇医药
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.
, 百拇医药
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
, 百拇医药
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.
, 百拇医药
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 (Hewlett Packard Company,Greeley,Colorado) with Scion Image program.The semi-quantified EMT expression for each cell line was determined by the reading from EMT divided by the reading from β-actin.
, 百拇医药
1.3 Western blot analysis
DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) and one of the nucleotide excision repair (NER) gene,ERCC2 protein expression was detected by Western blotting as previously described〔11〕.Briefly,total cellular proteins were extracted and separated by SDS-PAGE,electroblotted onto nitrocellulose, hybridized with primary antibody overnight at 4℃ (ERCC2 at 1∶ 5000 dilution or MGMT at 1∶ 500 dilution) followed by incubation with secondary antibody (sheep anti-mouse IgG at1 ∶ 1000 for ERCC2 or 1∶ 500 for MGMT) for one hour at 4℃ .Polyclonal ERCC2 primary antibody,MER-2,was raised against the whole recombinant ERCC2 protein in the mouse,a kind gift from Dr.Larry Thompson (Lawrence Livermore National Laboratory,Livermore, CA).The MGMT primary antibody,MT3.1, is a mouse monoclonal antibody (provided by Dr.thomas Brent at St.Jude Children's Research Hospital, Memphis,TN). Following ECL treatment and exposure to film,band intensities were quantitated by densitometric analysis (Ultroscan XL. LKB 2222-020). Similarly,α -tubulin expression was determined (primary mouse monoclonal IqG at 1∶ 1000,and secondary anti-mouse IgG at 1∶ 1000). For each cell line gene expression was normalized by dividing by α-tubulin.
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1.4 SRB cytotoxicity assay
The cytotoxicity of SarCNU was determined using a modified sulforhodamine B (SRB) colorimetric anticancer-drug screening assay〔11，12〕. Briefly, cells were seeded onto 24-well flat-bottom plates for 16 hours, washed once with PBS, and incubated for 60 minutes with different concentrations of SarCNU [in PBS supplemented with 0.7% BSA Fraction V,0.25% dextrose,0.001% phenol red pH 7.4,(PAG)].Following PAG aspiration,cells were fed with 2 ml medium and incubated at 37℃ ,5% CO2 for 7 days. The medium was then removed,and cells were fixed onto the plastic with 1 ml of 10% trichloroacetic acid (TCA) in 0.9% NaCl for one hour at 4℃ .Plates were washed five times with water to remove TCA,air dried,stained with l ml 0.4% SRB in 1% acetic acid for 30 minutes at room temperature,washed five times with 1% acetic acid to remove unbound dye, and subsequently air-dried.Bound dye was solubilized with 2 ml of 10 mM unbuffered Tris base (pH 10.5). Absorbance was read using a spectrophotometer at 500-550 nm,and IC90(μM)values were obtained by exponential curve fit of the linear portion of the cytotoxicity curve using CA-Cricket Graph III version 1.01 (Computer Associates International,Inc., Islandia,NY).
, 百拇医药
2 RESULTS
2.1 EMT expression in human tumor cell lines
Almost all of the cell lines examined were positive for EMT expression,although five cell lines (MGR-1,MGR-2,T98-G,SKI-1,and GBM) wer very low expressors (Fig.1). The semi-quantitative data of EMT expression is listed in Table 1, which is expressed as the mean of three separate experiments.Human hepatoma cell line HepG2 is a strong EMT expressor and is used as a positive control.
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2.2 Western blot result
ERCC2 and MGMT protein expression in the cell lines varied wildly.The value listed in Table l is expressed as the mean of three separate experiments for each cell line.The level of ERCD2 for each cell line is divided by ERCC2 protein level for SKNSH, for each experiment.MGMT protein level for each cell line is divided by MGMT protein level for MCF-7,for each experiment.
, 百拇医药
Figure 1. Human EMT expression in 23 human
tumor cell lines.A 1% agarose gel show the
333bp EMT PCR product and the 315 bp β-actin
PCR product.
2.3 Cytotoxicity of SarCNU
Cytotoxicity ot SarCNU is expressed as IC90(μM) and is the mean value of at least 3 repeated experiments (Table 1).
, 百拇医药
Table 1. Cytotoxicity of SarCUN,and ERCC-2,MGMT and
EMT gene expression in 18 human tumor cell lines cell line
(Tumor type)
SarCNU Cytotoxicity
(IC90)
Western Blot Results RT -PCR
ERCC2
MGMT
EMT
, 百拇医药
MCF-7(B)※
218.5
0.006
1
0.450
SKNSH(N)
367.7
1.000
0167
0.113
T98G(G)
165.2
, 百拇医药
0.065
0.306
0.020
A498(R)
451.6
0.316
0.130
1.147
HT-29(C)
80.7
0.002
0.613
0.958
, 百拇医药
caki-1(R)
284.8
0.093
0.394
0.913
ACHN(R)
146.2
0.182
0.680
0.653
SF-295(G)
46.5
, 百拇医药 0.051
0.037
0.185
skmg-1(G)
28.6
0.006
0
0.739
Skmg-4(G)
40.6
0.065
0.052
0.631
, 百拇医药
Ski-1(G)
44.8
0.050
0.012
0.053
MGR-3(G)
266.4
0.022
0.022
0.572
uw28(G)
62.2
0.018
, 百拇医药
0.134
0.524
786-0(R)
36.7
0.062
0.023
0.198
MGR-1(G)
72.4
0.009
0
0.050
MGR-2(G)
, 百拇医药
36.8
0.019
0
0.081
SW-620(C)
34.0
0.005
0.015
0.696
UWR-7(G)
33.4
0.188
0
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0.470
※ Specifies the type of tumor cell line： B,breast;C,colon;G,glioma;N, neuroblastoma;R,renal.
2.4 Comparison of EMT expression with cytotoxicity of SarCNU
Regression analysis was used to assess the relationship between SarCNU cytotoxicity and ERCC2protein,MGMT protein or EMT RNA levels.There was no significant linear correlation between SarCNU cytotoxicity and EMT expression. However,a significant correlation between SarCNU cytotoxicity and ERCC2 protein levels was found, corroborated previous findings of a relation between 1,3-bis (-chloroethyl)-1-nitroso-urea (BCNU),a standard CENU, cytotoxicity and ERCC2 protein levels〔11〕. Interestingly,multiple regression analysis rendered the correlation more significant as comparing SarCNU cytotoxicity with EMP plus ERCC2 and MGMT (Table 2).
, 百拇医药
Table 2. Correlation between SarCNU cytotoxicity and
gene expression in 18 human tumor cell lines SarCNU
-ERCC2
0.596
0.0098
-MGMT
0.293
0.2381
-EMT
0.402
, 百拇医药
0.0985
SarCNU
-ERCC2+MGMT
0.666
0.0124
-ERCC2+EMT
0.727
0.0036
-MGMT+EMT
0.482
0.1373
SarCNU
, 百拇医药
-EMT+MGMT+ERCC2
0.779
0.0037
3 DISCUSSION
Nitrosoureas, such as BCNU and 1-(2-chloro-ethyl)-3-cyclohexyl-1-nitrosourea (CCNU), have long been used as the standard chemotherapeutic compounds for the treatment of central nervous system (CNS) tumors〔13〕.Unfortunately,in most patients,these drugs do not produce durable long-term responses even in combination with radiation therapy〔14〕. In search for novel analogues with increased antitumor activity and decreased toxicity,SarCNU was found to have interesting characteristics〔3〕. SarCNU contains an amino acid amide group〔15〕,N-methylglycinamide,known as sarcosinamide, which allows the drug to enter cells via the extraneuronal monoamine transporter.
, 百拇医药
Our previous in vitro studies demonstrated that SarCNU was more effective than BCNU against human gliomas〔5,6〕. Using transport studies with radiolabeled SarCNU we have demonstrated that the uptake of SarCNU in SKMG-1 cells were more than that in SKI-1 cells〔4〕, which corresponded to the cytotoxicity of SarCNU to SK-MG-1 cells (IC90=28.6 μM) and SKI-1 (IC90=44.8 μM). Utilizing RT-PCR, we confirmed in this study,that SKI-1 cells were EMT-poor whereas SKMG-1 cells were EMT-rich,supporting that the differential cytotoxicity to SarCNU in these cell lines is attributed to the presence of the EMT in SKMG-1 cells.In in vivo studies〔7,8〕,we evaluated the antitumor activity of SarCNU with the human glioma xenografts SF-295, U-251 and SHG-44.SarCNU was more effective than BCNU against these tumors,which have been confirmed EMT positive,suggesting that EMT is important in the in vivo response to SarCNU.
, 百拇医药
It has been documented by both laboratory and clinical evidence that MGMT plays an important role in CENU drug resistance〔16～19〕. We also demonstrated that NER,specifically,ERCC2 related to CENU resistance in human tumor cell lines〔12，20，213〕, as also revealed in the present study,ERCC2 protein levels significantly correlated to SarCNU cytotoxicity.In the present investigation,the absence of a linear correlation between SarCNU cytoxoxicity and EMT expression in these human tumor cell lines may be due, at least in part,to the presence of DNA repair factors such as MGMT and ERCC2.This is suggested by the multiple regression analysis,that EMT enhanced correlation between SarCNU cytotoxicity and ERCC2 or ERCC2 plusMGMT expression.Thus,for tumor cells with similar levels of such DNA repair proteins,the presence of the EMT may be a determining factor in responsiveness to SarCNU.
, 百拇医药
This panel of 23 human tumor cell lines of different origins shows that the majority (about 80% ) express the EMT. Assuming that human tumor cell lines are reflective of the clinical situation, SarCNU should prove to be a more extend effective alternative chemotherapeutic agent for treatment of human tumors.The presence of EMT could be used to identify cancer patients who may be potential responders to SarCNU in the clinic.This bears direct clinical relevance since SarCNU has recently entered phase I clinical trialls.
, 百拇医药
Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, McGill University,3755 Cote Ste Catherine,Montreal Quebec,H3T 1E2,Canada
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, 百拇医药
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〔7〕 Marcantonio D,Panasci LC,Hollingshead MG,et al. SarCNU, a novel chloroethylnitrosourea analogue with enhanced antitumor activity against human glioma xenografts〔J〕. Cancer Res,1997,57：3895～3898.
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〔10〕 Chen Z-P,Malapetsa A,Mohr G,et al. Quantitation of ERCC-2 gene expression in human tumor cell lines by reverse transcription-polymerase chain reaction in comparison to northern blot analysis〔J〕. Anal Biochem,1997,244：50～54.
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〔15〕 Suami T,Kato T,Takino H,et al. (2-Chloroethyl)nitrosourea congeners of amino acid amides〔J〕. J Med Chem, 1982,25：829～832.
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〔18〕 Jaeckle KA,Eyre HJ,Townsend JJ,et al. Correlation of tumor O6-methylguanine-DNA methyltransferase levels with survival of malignant astrocytoma patients treated with bis-chloroethylnitroso-urea：a Southwest Oncology Group study〔J〕. J Clin Oncol, 1998,16：3310～3315.
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收稿日期：1999-06-02, 百拇医药
单位：中山医科大学肿瘤防治中心脑外科(广州，510060)
关键词：神经元外单胺转运蛋白；2-氯乙基-3-肌氨酸胺-1-亚硝基脲；人肿瘤细胞系
癌症990505
【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。
, http://www.100md.com
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 Mohr Lawrence C. Panasci
Lady Davis Institute for Medical Research,Sir Mortimer B.Davis-Jewish General Hospital,McGill University,3755 C
te Ste Catherine,Montreal,Quebec,H3T 1E2,Canada, 百拇医药
【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.
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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.
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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
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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.
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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 (Hewlett Packard Company,Greeley,Colorado) with Scion Image program.The semi-quantified EMT expression for each cell line was determined by the reading from EMT divided by the reading from β-actin.
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1.3 Western blot analysis
DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) and one of the nucleotide excision repair (NER) gene,ERCC2 protein expression was detected by Western blotting as previously described〔11〕.Briefly,total cellular proteins were extracted and separated by SDS-PAGE,electroblotted onto nitrocellulose, hybridized with primary antibody overnight at 4℃ (ERCC2 at 1∶ 5000 dilution or MGMT at 1∶ 500 dilution) followed by incubation with secondary antibody (sheep anti-mouse IgG at1 ∶ 1000 for ERCC2 or 1∶ 500 for MGMT) for one hour at 4℃ .Polyclonal ERCC2 primary antibody,MER-2,was raised against the whole recombinant ERCC2 protein in the mouse,a kind gift from Dr.Larry Thompson (Lawrence Livermore National Laboratory,Livermore, CA).The MGMT primary antibody,MT3.1, is a mouse monoclonal antibody (provided by Dr.thomas Brent at St.Jude Children's Research Hospital, Memphis,TN). Following ECL treatment and exposure to film,band intensities were quantitated by densitometric analysis (Ultroscan XL. LKB 2222-020). Similarly,α -tubulin expression was determined (primary mouse monoclonal IqG at 1∶ 1000,and secondary anti-mouse IgG at 1∶ 1000). For each cell line gene expression was normalized by dividing by α-tubulin.
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1.4 SRB cytotoxicity assay
The cytotoxicity of SarCNU was determined using a modified sulforhodamine B (SRB) colorimetric anticancer-drug screening assay〔11，12〕. Briefly, cells were seeded onto 24-well flat-bottom plates for 16 hours, washed once with PBS, and incubated for 60 minutes with different concentrations of SarCNU [in PBS supplemented with 0.7% BSA Fraction V,0.25% dextrose,0.001% phenol red pH 7.4,(PAG)].Following PAG aspiration,cells were fed with 2 ml medium and incubated at 37℃ ,5% CO2 for 7 days. The medium was then removed,and cells were fixed onto the plastic with 1 ml of 10% trichloroacetic acid (TCA) in 0.9% NaCl for one hour at 4℃ .Plates were washed five times with water to remove TCA,air dried,stained with l ml 0.4% SRB in 1% acetic acid for 30 minutes at room temperature,washed five times with 1% acetic acid to remove unbound dye, and subsequently air-dried.Bound dye was solubilized with 2 ml of 10 mM unbuffered Tris base (pH 10.5). Absorbance was read using a spectrophotometer at 500-550 nm,and IC90(μM)values were obtained by exponential curve fit of the linear portion of the cytotoxicity curve using CA-Cricket Graph III version 1.01 (Computer Associates International,Inc., Islandia,NY).
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2 RESULTS
2.1 EMT expression in human tumor cell lines
Almost all of the cell lines examined were positive for EMT expression,although five cell lines (MGR-1,MGR-2,T98-G,SKI-1,and GBM) wer very low expressors (Fig.1). The semi-quantitative data of EMT expression is listed in Table 1, which is expressed as the mean of three separate experiments.Human hepatoma cell line HepG2 is a strong EMT expressor and is used as a positive control.
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2.2 Western blot result
ERCC2 and MGMT protein expression in the cell lines varied wildly.The value listed in Table l is expressed as the mean of three separate experiments for each cell line.The level of ERCD2 for each cell line is divided by ERCC2 protein level for SKNSH, for each experiment.MGMT protein level for each cell line is divided by MGMT protein level for MCF-7,for each experiment.
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Figure 1. Human EMT expression in 23 human
tumor cell lines.A 1% agarose gel show the
333bp EMT PCR product and the 315 bp β-actin
PCR product.
2.3 Cytotoxicity of SarCNU
Cytotoxicity ot SarCNU is expressed as IC90(μM) and is the mean value of at least 3 repeated experiments (Table 1).
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Table 1. Cytotoxicity of SarCUN,and ERCC-2,MGMT and
EMT gene expression in 18 human tumor cell lines cell line
(Tumor type)
SarCNU Cytotoxicity
(IC90)
Western Blot Results RT -PCR
ERCC2
MGMT
EMT
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MCF-7(B)※
218.5
0.006
1
0.450
SKNSH(N)
367.7
1.000
0167
0.113
T98G(G)
165.2
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0.065
0.306
0.020
A498(R)
451.6
0.316
0.130
1.147
HT-29(C)
80.7
0.002
0.613
0.958
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caki-1(R)
284.8
0.093
0.394
0.913
ACHN(R)
146.2
0.182
0.680
0.653
SF-295(G)
46.5
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0.037
0.185
skmg-1(G)
28.6
0.006
0
0.739
Skmg-4(G)
40.6
0.065
0.052
0.631
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Ski-1(G)
44.8
0.050
0.012
0.053
MGR-3(G)
266.4
0.022
0.022
0.572
uw28(G)
62.2
0.018
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0.134
0.524
786-0(R)
36.7
0.062
0.023
0.198
MGR-1(G)
72.4
0.009
0
0.050
MGR-2(G)
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36.8
0.019
0
0.081
SW-620(C)
34.0
0.005
0.015
0.696
UWR-7(G)
33.4
0.188
0
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0.470
※ Specifies the type of tumor cell line： B,breast;C,colon;G,glioma;N, neuroblastoma;R,renal.
2.4 Comparison of EMT expression with cytotoxicity of SarCNU
Regression analysis was used to assess the relationship between SarCNU cytotoxicity and ERCC2protein,MGMT protein or EMT RNA levels.There was no significant linear correlation between SarCNU cytotoxicity and EMT expression. However,a significant correlation between SarCNU cytotoxicity and ERCC2 protein levels was found, corroborated previous findings of a relation between 1,3-bis (-chloroethyl)-1-nitroso-urea (BCNU),a standard CENU, cytotoxicity and ERCC2 protein levels〔11〕. Interestingly,multiple regression analysis rendered the correlation more significant as comparing SarCNU cytotoxicity with EMP plus ERCC2 and MGMT (Table 2).
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Table 2. Correlation between SarCNU cytotoxicity and
gene expression in 18 human tumor cell lines SarCNU
-ERCC2
0.596
0.0098
-MGMT
0.293
0.2381
-EMT
0.402
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0.0985
SarCNU
-ERCC2+MGMT
0.666
0.0124
-ERCC2+EMT
0.727
0.0036
-MGMT+EMT
0.482
0.1373
SarCNU
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-EMT+MGMT+ERCC2
0.779
0.0037
3 DISCUSSION
Nitrosoureas, such as BCNU and 1-(2-chloro-ethyl)-3-cyclohexyl-1-nitrosourea (CCNU), have long been used as the standard chemotherapeutic compounds for the treatment of central nervous system (CNS) tumors〔13〕.Unfortunately,in most patients,these drugs do not produce durable long-term responses even in combination with radiation therapy〔14〕. In search for novel analogues with increased antitumor activity and decreased toxicity,SarCNU was found to have interesting characteristics〔3〕. SarCNU contains an amino acid amide group〔15〕,N-methylglycinamide,known as sarcosinamide, which allows the drug to enter cells via the extraneuronal monoamine transporter.
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Our previous in vitro studies demonstrated that SarCNU was more effective than BCNU against human gliomas〔5,6〕. Using transport studies with radiolabeled SarCNU we have demonstrated that the uptake of SarCNU in SKMG-1 cells were more than that in SKI-1 cells〔4〕, which corresponded to the cytotoxicity of SarCNU to SK-MG-1 cells (IC90=28.6 μM) and SKI-1 (IC90=44.8 μM). Utilizing RT-PCR, we confirmed in this study,that SKI-1 cells were EMT-poor whereas SKMG-1 cells were EMT-rich,supporting that the differential cytotoxicity to SarCNU in these cell lines is attributed to the presence of the EMT in SKMG-1 cells.In in vivo studies〔7,8〕,we evaluated the antitumor activity of SarCNU with the human glioma xenografts SF-295, U-251 and SHG-44.SarCNU was more effective than BCNU against these tumors,which have been confirmed EMT positive,suggesting that EMT is important in the in vivo response to SarCNU.
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It has been documented by both laboratory and clinical evidence that MGMT plays an important role in CENU drug resistance〔16～19〕. We also demonstrated that NER,specifically,ERCC2 related to CENU resistance in human tumor cell lines〔12，20，213〕, as also revealed in the present study,ERCC2 protein levels significantly correlated to SarCNU cytotoxicity.In the present investigation,the absence of a linear correlation between SarCNU cytoxoxicity and EMT expression in these human tumor cell lines may be due, at least in part,to the presence of DNA repair factors such as MGMT and ERCC2.This is suggested by the multiple regression analysis,that EMT enhanced correlation between SarCNU cytotoxicity and ERCC2 or ERCC2 plusMGMT expression.Thus,for tumor cells with similar levels of such DNA repair proteins,the presence of the EMT may be a determining factor in responsiveness to SarCNU.
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This panel of 23 human tumor cell lines of different origins shows that the majority (about 80% ) express the EMT. Assuming that human tumor cell lines are reflective of the clinical situation, SarCNU should prove to be a more extend effective alternative chemotherapeutic agent for treatment of human tumors.The presence of EMT could be used to identify cancer patients who may be potential responders to SarCNU in the clinic.This bears direct clinical relevance since SarCNU has recently entered phase I clinical trialls.
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Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, McGill University,3755 Cote Ste Catherine,Montreal Quebec,H3T 1E2,Canada
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收稿日期：1999-06-02, 百拇医药