反义寡核苷酸并超声微泡造影剂转染联合超声照射逆转肝癌多药耐药

目的探讨体内、体外mdr1、mrp、lrp反义寡核苷酸(AODNs)并超声微泡造影剂转染联合低强度超声照射逆转肝癌多药耐药的可行性,寻找逆转肿瘤多药耐药有效和靶向的方法。方法利用超声微泡造影剂包载肿瘤耐药基因mdr1、mrp、lrp的AODNs进行转染,联合低强度超声照射,以肝癌细胞多药耐药细胞模型(HepG2／ADM) 为研究对象,通过逆转录聚合酶链反应、western blot和四甲基偶氮唑盐法,从体外细胞培养及动物实验,研究 AODNs并超声微泡造影剂转染联合超声照射逆转癌细胞多药耐药及降低肿瘤恶性表型和成瘤能力的作用。结果 HepG2／AMD细胞增殖被抑制,其mdr1和mrp的mRNA、蛋白质表达水平明显降低;裸鼠皮下移植瘤生长受抑制。结论体外、体内AODNs并超声微泡造影剂转染联合低强度超声照射能有效逆转人肝癌细胞HepG2／ADM的多药耐药, 该技术可能为肝癌临床治疗提供新的思路。     

携多药耐药基因1反义RNA重组腺病毒逆转肝癌多药耐药细胞的实验研究

目的探讨携带多药耐药基因1(multidrug resistance gene 1,MDR1)反义RNA重组腺病毒载体在裸鼠体内逆转肝癌多药耐药细胞HepG2/阿霉素(adriamycin,ADM)的疗效及作用机制。方法构建携带AFP和asmdr1的重组腺病毒载体Adeno-asmdr1,ADM分级诱导肝癌细胞HepG2为多药耐药细胞HepG2/ADM,建立HepG2/ADM裸鼠皮下移植瘤模型,Adeno asmdr1局部注射,观察移植瘤的体积、透射电镜检测移植瘤组织细胞凋亡、RT-PCR检测MDR1转录水平,评价Adeno-asmdr1的抗肿瘤活性。结果在Adeno-asmdr1 ADM组,移植瘤体积无增大,而PBS组、ADM组体积明显增大;RT-PCR检测移植瘤细胞1周和4周MDR1 mRNA水平, ADM组无明显变化,Adeno-amdr1 ADM组在4周时MDR1转录水平仅为单纯ADM组的20%,经ADM Adeno-asmdr1处理组,可见凋亡增加,ADM组和PBS处理组的裸鼠移植瘤组织中出现少量或没有凋亡。结论携带MDR1反义RNA重组腺病毒部分逆转HepG2/ADM的多药耐药,阻止肿瘤生长,下调MDR1转录水平,导致肿瘤细胞凋亡。     

Subcellular daunorubicin distribution and its relation to multidrug resistance phenotype in drug—resistant cell line SMMC—7721／R

AIM：To investigate the correlation between subcellular daunorubicin distribution and the multidrug resistance phenotype in drug-resistant cell line SMMC-7721/R.METHODS:The multidrug resistant cell line SMMC-7721/R,a human hepatocellular carcinoma cell line,was established.Antisenes oligonucleotides(AS-ODN)were used to obtain different multidrug resistance phenotypes by inhibiting the expression of mdr1 gene and/or multidrug resistance-related protein gene(mrp)using Lipofectamine as delivery agent.Expression of mdr1 and mrp genes was evaluated by RT-PCRand Western blotting.Intracellular daunorubicn(DNR)concentration was measured by flow cytometry.Subcellular DNR distribution was analyzed by confocal laser scanning microscopy.Adriamycin(ADM)and DNR sensitivity was examined by MTTmethod.RESULTS:Low level expression of mdr1 and mrpmRNAs and no expression of P-Glycoprotein(P-gp)and multidrug resistance-related protein(P190)were detected in parental sensitive cells SMMC-7721/S,but over-expression of these two genes was observed in drug-resistant cell SMMC-7721/R,The expression of mdr1 and mrp genes in SMMC-7721/Rcells was down-regulated to the level in the SMMC-7721/Scells by AS-ODN.Intracellular DNAconcentration in SMMC-7721/Scells was 10times higher than that in SMMC-7721/Rcells.In SMMC7721/Scells intracellular DNA distributed evenly in the nucleus and cytoplasm.while in SMMC-7721/Rcells DNR distributed in a punctate pattern in the cytoplasm and was reduced in the nucleus.DNR concentration in SMMC-7721/Rcells co-transfected with AS-ODNs targeting to mdr1and rpmRNAs recovered to 25percent of that in SMMC7721/Scells.Intracellular DNA distribution pattern in drug-resistant cells treated by AS-ODN was similar to drug-sensitive cell.and the cells resistance index(RI)to DNA and AMD decreased at most from 88.0and 116.0to4.0and 2.3,repectively.Co-Transfection of two AS-ODNs showed a stronger synergistic effect than separate transfection.CONCLUSIONS:P-gp and P190are two members mediatingMDR in cellline SMMC7721/R,Intracellular drug concentration increase and subcellular distribution change are two important factos in multidrug resistance(MDR)formation.The second facto,drugs transport by P-gp andP190from cell nucleus to organell in cytoplasm,may play a more important role.     

Reversal of multi-drug resistance by pSUPER-shRNA-mdr1 in vivo and in vitro

AIM： To explore the possibility of reversing multi-drug resistance （MDR） to HepG2/mdr1 in vitro and in vivo with RNA interference （RNAi）. METHODS： HepG2/mdr1 was obtained by cloning the whole gene mdrl into HepG2 cells, shRNA targeting sequence was designed to be homologous to the P-gp encoding MDR1 mRNA consensus sequence, pSUPER- shRNA/mdrl was constructed using the enzyme- digested technique. HepG2/mdrl cells were transfected with vectors of pSUPER-shRNA/mdrl to measure their efficacy by real-time PCR for mdrl mRNA, flow cytometry （FCM） for P-gp expression, and Rhodamine efflux, MTT method for HepG2/mdrl function, respectively. In vivo, mice tumors were treated by injecting pSUPER-shRNA/mdrl in situ and into intra- abdominal cavity. Tumors were collected to create cell suspension and cryosections after chemothearpy with adiramycin and mytomycin. The cell suspension was incubated in RPMI-1640 supplemented with G418 to screen stable cells for appreciating the reversal of MDR. Cryosections were treated with immunohistochemistry technique to show the effectiveness of transfection and the expression of P-gp.RESULTS： pSUPER-shRNA/mdrl was successfully constructed, which was confirmed by sequencing. The MDR phenotype of HepG2/mdr1 was decreased significantly in vitro transfection. HepG2/mdr1 showing its MDR was reversed notably in P-gp expression （11.0% vs 98.2%, P 〈 0.01）. Real-time PCR showed that mRNA/mdrl was lower in test groups than in control groups （18.73± 1.33 vs 68.03±2.21, P 〈 0.001）. Compared with HepG2, the sensitivity of HepG2/ mdrl and HepG2/mdr1-dsRNA cells to ADM was decreased by 1.64 times and 15.6 times, respectively. The accumulation of DNR in positive groups was decreased evidently. In vivo, the p-gp expression in positive groups was significantly lower than that in control groups （65.1% vs 94.1%, P 〈 0.05）. The tumor suppressing rate in test groups was 57.8%. After chemotherapy, the growth rate in test groups was lower than that in control groups （700.14 ± 35.61 vs 1659.70 ± 152.54, P 〈 0.05）. Similar results were also observed under fluorescence microscope, and confirmed by Image-Pro Plus 4.5 analysis. CONCLUSION： pSUPER-shRNA/mdrl vector system allows simple, stable and durable nonviral knockdown of P-gp by RNAi in malignant cells and animals to restore their sensitivity to adriamycin.     

Down-regulation of extracellular signal-regulated kinase 1/2 activity in P-glycoprotein-mediated multidrug resistant hepatocellular carcinoma cells

AIM： To study the expression and phosphorylation of extracellular signal-regulated kinase （ERK） i and ERK2 in multidrug resistant （MDR） hepatocellular carcinoma （HCC） cells.
METHODS： MDR HCC cell lines, HepG2/adriamycin （ADM） and SMMC7721/ADM, were developed by exposing parental cells to stepwise increasing concentrations of ADM. MTT assay was used to determine drug sensitivity. Flow cytometry was employed to analyze cell cycle distribution and measure cell P-glycoprotein （P-gp） and multidrug resistant protein 1 （MRP1） expression levels. ERK1 and ERK2 mRNA expression lev-ls were measured by quantitative real-time PCR （QRTPCR）. Expression and phosphorylation of ERK1 and ERK2 were analyzed by Western blot.
RESULTS： MTT assay showed that HepG2/ADM andSMMC7721/ADM were resistant not only to ADM, but also to multiple anticancer drugs. The P-gp expression was over 10-fold higher in HepG2/ADM cells than in HepG2 cells （8.92% ±0.22% vs 0.88% ± 0.05%, P 〈 0.001） and over 4-fold higher in SMMC7721/ADM cells than in SMMC7721 cells （7.37% ± 0.26% vs 1.74% ± 0.25%, P 〈 0.001）. However, the MRP1 expression was not significantly higher in HepG2/ADM and SMMC7721/ADM cells than in parental cells. In addition, the percentage of MDR HepG2/ADM and SMMC7721/ADM cells was significantly decreased in the G0/G1 phase and increased in the the S phase or G2/M phase. QRT-PCR analysis demonstrated that the ERK1 and ERK2 mRNA expression increased apparently in HepG2/ADM cells and decreased significantly in SMMC7721/ADM cells. Compared with the expression of parental cells, ERK1 and ERK2 protein expressions were markedly decreased in SMMC7721/ADM cells. However, ERK2 protein expression was markedly increased while ERK1 protein expression had no significant change in HepG2/ADM cells. Phosphorylation of ERK1 and ERK2 was markedly decreased in both HepG2/ADM and SMMC7721/ADM MDR cells.
CONCLUSION： ERK1 and ERK2 activities are downregulated in P-gp-mediated MDR HCC cells. ERK1 or ERK2 might be a potential drug target for circumventing MDR HCC cells,     

三氧化二砷逆转肝癌细胞株HepG2/ADM多药耐药的作用

目的:探讨三氧化二砷(As2O3)体外逆转人肝癌细胞多药耐药性的作用及机制．方法:MTT法检测As2O3的细胞毒作用和处理前后耐药细胞对化疗药物的敏感性,用流式细胞仪检测细胞内阿霉素浓度,通过RT-RCR检测MDR1基因的表达．结果:As2O3在0．25 mg／L剂量以下时对HepG2和HepG2／ADM耐药细胞株的抑制率均小于15％,半数抑制率(IC50)分别为1．02和1．34 mg／L,无细胞毒剂量0．2 mg/L的As2O3能部分逆转HepG2／ADM细胞对阿霉素、顺铂(CDDP)、丝裂霉素(MMC)、5-氟尿嘧啶(5-FU)的耐药性,逆转倍数分别为2．92,3．09,2．13,2．60倍．同时无细胞毒剂量0．2 mg／L的As2O3能使HepG2／ADM细胞内阿霉素浓度明显增加,MDR1表达下降．结论:As2O3具有体外逆转人肝癌细胞多药耐药性的作用,可能与下调MDR1表达、增加细胞内药物积累有关．     

Increase of doxorubicin sensitivity by doxorubicin-loading into nanoparticles for hepatocellular carcinoma cells in vitro and in vivo

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is known to be chemoresistant to anticancer drugs due to the multidrug resistant (MDR) transporters expression. Here, we compared in vitro and in vivo the anti-tumor efficacy of doxorubicin-loaded polyisohexylcyanoacrylate nanoparticles (PIHCA-Dox) versus free doxorubicin (Dox). These nanoparticles are known to overcome the MDR phenotype. METHODS: We first determined in vitro the 50% inhibition concentration (IC(50)) of these drugs on different human hepatoma cell lines. Secondly, the efficacy of the drugs in vivo was determined on the X/myc transgenic murine model of HCC by histological counting of apoptotic tumorous hepatocytes and by TUNEL labeling. We characterized by semi-quantitative RT-PCR the MDR-related gene (mdr1, mdr3, mrp1) expression pattern in this model. RESULTS: In vitro, IC(50) was reduced with PIHCA-Dox versus Dox for Huh7 (1.7-fold reduction; P<0.001), HepaRG (4.5-fold reduction; P<0.01), HepG2 (1.5-fold reduction; P<0.001), and HepG2.2.15 (1.5-fold reduction; P=0.059). In vivo, HCC in transgenic mice overexpressed the mdr1 and mdr3 genes and the antitumor drugs efficacy was greatly enhanced after injection of PIHCA-Dox (9.0+/-5.0%; n=15) versus Dox (4.6+/-3.3%; n=13; P=0.01) for apoptotic bodies count. CONCLUSIONS: These promising data showing a higher anti-tumor efficacy on HCC of PIHCA-Dox versus Dox, warrant further studies in both animals and humans.     

Reversing multidrug resistance by RNA interference through the suppression of MDR1 gene in human hepatoma cells

AIM: To reverse the multidrug resistance (MDR) by RNA interference (RNAi)-mediated MDR1 suppression in hep-atoma cells. METHODS: For reversing MDR by RNAi technology, two different short hairpin RNAs (shRNAs) were designed and constructed into pGenSil-1 plasmid, respectively. They were then transfected into a highly adriamycin-resistant HepG2 hepatoma cell line (HepG2/ADM). The RNAi effect on MDR was evaluated by real-time PCR, cell cytotoxicity assay and rhodamine 123 (Rhl23) efflux assy. RESULTS: The stably-transfected clones showed various degrees of reversal of MDR phenotype. Surprisingly, the MDR phenotype was completely reversed in two transfected clones. CONCLUSION: MDR can be reversed by the shRNA-mediated MDRI suppression in HepG2/ADM cells, which provides a valuable clue to make multidrug-resistant hepatoma cells sensitive to anti-cancer drugs.     

Effects of multidrug resistance, antisense RNA on the chemosensitivity of hepatocellular carcinoma cells

Introduction The overexpression of the multidrug resistance gene 1 (mdr1), a product of multidrug resistance multidrug resistance, is a major obstacle in cancer chemotherapy. Being a major P-glycoproteincancer chemotherapy. Being a major P-glycoprotein (P-gp), 17 kDa transmembrane protein acts as an energy-dependent drug efflux pump and keeps the concentration and efficacy intracellular anticancer drugs low.[1-4] Hepatocellular carcinoma (HCC) represents more than 5% of all cancers in the w…     

Combined effects of Cantide and chemotherapeutic drugs on inhibition of tumor cells＇ growth in vitro and in vivo

AIM: To investigate the combination effect of hTERT antisense oligonucleotide "Cantide" and three chemotherapeutic drugs (cisplatin, 5-fluorouracil (5-FU) and adriamycin (ADM)) on inhibiting the proliferation of HepG2, BGC and A549 cell lines in vitro, and to investigate the efficacy of Cantide used in combination with cisplatin (DDP) in vivo. METHODS: Cantide was transfected into these tumor cells by Lipofectin, and cell growth activity was calculated by microcytotoxicity assay. In vivo study, cells of HepG2 were implanted in Balb/c nude mice for 4 d. Then Cantide, DDP and Cantide+DDP were given intra peritonea Ily for 24 d respectively. The body weights of the tumor-bearing animals and their tumor mass were measured later to assess the effect of combination therapy in the nude mice. To evaluate the interaction of Cantide and these chemotherapeutic drugs, SAS software and Jin Zhengjun method were used. RESULTS: Combination treatments with 0.1μmol/L Cantide reduced the IC50 of DDP, 5-FU and ADM from 1.07, 4.15 and 0.29 μg/mL to 0.25,1.52 and 0.12 μg/mL respectively. The inhibition ability of DDP, 5-FU and ADM respectively in combination with Cantide in these tumor cells was higher than that of these drugs alone (P<0.0001). And synergism (Q≥1.15)was observed at the lower concentration of DDP (≤1μg/mL) and ADM (≤0.1 μg/mL) with combination of Cantide. In vivo, combination treatment with Cantide and DDP produced the greater growth inhibition of human liver carcinoma cells HepG2 in nude mice (0.65±0.19 g tumor) compared with that when only one of these drugs was used (Cantide group: 1.05±0.16 g tumor, P= 0.0009<0.001; DDP group: 1.13±0.09 g tumor, P= 0.0001<0.001). CONCLUSION: These findings indicate that Cantide may enhance therapeutic effectiveness of chemotherapeutic drugs over a wide range of tumor cells in vitro, and the combination use of Cantide and DDP can produce much higher inhibition rates, as compared with when either of these drugs was used only in vivo.     

高温对SGC7901/ADM细胞多药耐药蛋白表达的影响和意义

目的:研究高温43℃对多药耐药基因表达产物P-gp、MRP、LRP在蛋白水平上的影响及其生物学意义,更深入地了解热效应逆转耐药的机制.方法:采用免疫细胞化学染色法、RT-PCR以及Western blot方法,检测在不同温度和不同药物作用下,人胃癌耐药细胞株SGC7901/ADM和对照的人胃癌敏感细胞SGC7901株中,与人胃癌多药耐药相关的分子MDR1、P-gp、MRP、LRP在蛋白水平上的表达差异.结果:采用免疫细胞化学染色法检测人胃癌耐药株SGC7901/ADM细胞,发现高温43℃60 min处理可使P-gp蛋白表达下调率(down-regulated rate,DRR)31.78%(P=0.016),而MRP表达DRR为20.22%(P=0.037),差异有统计学意义,LRP未表达.采用Western blot法在SGC7901细胞中未检测出P-gp蛋白表达,而SGC7901/ADM细胞中P-gp高表达;在ADM和CDDP处理组细胞中,高温43℃时P-gp的表达量较37℃时DRR分别为45.65%(P=0.007)、17.95%(P=0.021),差异有显著学意义;TAX处理组DRR为11.90%,差异无显著学意义(P=0.065).结论:高温43℃的短期处理对人胃癌SGC7901/ADM细胞中P-gp和MRP多药耐药蛋白的表达有一定的抑制作用,可能是逆转耐药的机制之一.     

重组腺病毒载体携带多药耐药基因1反义RNA靶向逆转肝癌细胞多药耐药

目的 探讨携带多药耐药基因1(multidrug resistance gene 1,mdr1)反义RNA的重组腺病毒载体靶向逆转甲胎蛋白阳性(AFP+)的肝癌多药耐药细胞HepG2R的疗效及作用机制.方法 分别构建携带AFP启动子和mdr1基因反义核苷酸片段的重组腺病毒载体Adeno-asmdr及携带AFP启动子和增强绿色荧光蛋白基因的重组腺病毒载体Adeno-EGFP,将Adeno-EGFP转染人正常肝细胞L02(AFP-),人官颈癌细胞HeLa(AFP-)及HepG2(AFP+)细胞,检测增强绿色荧光蛋白基因在各细胞的转录水平;将Adeno-asmdr转染HepG2R细胞,Western blot检测不同时间P-gp170的表达,末端脱氧核苷酸转移酶介导的脱氧三磷酸尿苷缺口末端标记法检测HepG2R细胞凋亡,流式细胞术检测HepG2R细胞在不同药物作用下细胞周期、凋亡率.结果 增强绿色荧光蛋白基因在AFP阳性的HepG2细胞可得到显著转录,而在L02细胞和HeLa细胞,其转录减少,显示了该载体的良好转录活性以及靶向特异性.Adeno-asmdr转染HepG2R细胞后,HepG2R细胞P-gp170表达明显减弱,HepG2R细胞凋亡增加,HepG2R细胞对多种化疗药物的耐受能力明显下降,细胞出现显著的周期阻滞,大量细胞被阻滞于S期和G0/M期,凋亡细胞比例增加.结论 实验构建的Adneo-asmdr重组腺病毒载体可在AFP阳性HepG2R细胞内特异靶向性表达目的 基因,并可有效降低mdrl基因产物P-gp170的表达,从而达到对HepG2R细胞多药耐药的逆转作用.     

溴隐亭逆转肝癌多药耐药的机制

目的探讨溴隐亭对肝癌多药耐药逆转的作用机制。方法体外实验分亲本细胞HepG2组（A组），耐药细胞HepG2／ADM组（B组）和B组加溴隐亭称为C组。分别检测各组细胞内荧光强度变化，细胞P-糖蛋白、蛋白激酶C—α蛋白表达情况，及5种肿瘤药物半数抑制浓度及耐药指数变化。分别将肝癌细胞HepG2和耐药细胞HepG2／ADM原位种植入裸鼠肝脏内，称A组鼠和B组鼠，另设B组种植鼠给予溴隐亭灌胃治疗称C组鼠。B超观察种植瘤的生长情况，肿瘤大小为1．0cm左右时经腹腔化疗，2周后处死裸鼠计算肿瘤的体积和质量抑制率，观察种植瘤组织多药耐药基因1不敷出mRNA表达情况，及治疗后肝癌细胞的凋亡指数。单独检测24只种植耐药细胞HepG2／ADM的裸鼠在服用溴隐亭前后分别注射的^99mTc—MIBI在肝脏肿瘤部位的潴留情况。结果体外实验结果显示在溴隐亭浓度为10μmol／L时罗丹明123在细胞内的潴留率均明显增加，且呈时间依赖性，对阿霉素的耐药逆转以溴隐亭浓度为10μmol／L时最显著，逆转率为82．6%。蛋白激酶C—α蛋白表达，C组与B组比较差异有统计学意义（q=5．37，P〈0．01），但两组P-糖蛋白表达差异无统计学意义（q=1．86，P〉0．05）。体内试验结果显示种植瘤的体积和质量抑制率比较，C组鼠明显高于B组鼠（q1=5．89，Q=4．92，P〈0．01），接近于A组鼠（功=2．47，q2=3．02，P〉0．05）。C组鼠与B组鼠多药耐药基因1mRNA表达差异无统计学意义（q=3．71，P〉0．05）。化疗后肿瘤细胞凋亡率比较，C组鼠明显高于B组鼠（q=3．72，P〈0．01）。口服溴隐亭后肝肿瘤组织对^99mTc-MIBI的潴留指数明显提高（t=3．58，P〈0．01）。结论溴隐亭通过抑制P-糖蛋白的功能可有效的逆转肝癌多药耐药性。     

肿瘤坏死因子相关凋亡诱导配体对肝癌耐药细胞株阿霉素化疗敏感性的影响

目的 研究肿瘤坏死因子相关凋亡诱导配体(TRAIL)联合阿霉素(ADM)处理肝癌耐药细胞株HePG2／ADM对化疗敏感性的影响。方法 通过培养液中ADM的浓度梯度增加法长期筛选培养，建立肝癌HepG2／ADM耐药细胞株，荧光定量PCR检测多药耐药(MDR)1的表达，TRAIL联合化疗药物ADM处理HePG2／ADM，MTT比色法检测细胞增殖，细胞凋亡采用流式细胞仪和TUNEL法检测观察HePG2／ADM对化疗药物的敏感性变化。结果 HepG2／ADM细胞是一个明确的多药耐药细胞模型，联合TRAIL(100ng／L) ADM(0．1mg／L)后，MTT显示HepG2／ADM细胞的增殖明显抑制，流式细胞术、TUNEL法检测TRAIL联合ADM处理HePG2／ADM细胞诱导的凋亡，与对照组相比，凋亡指数显著增加。结论 MDR1不参与TRAIL耐受。TRAIL可部分逆转HepG2／ADM对ADM的耐药，增加其对化疗药物的敏感性。联合TRAIL和亚毒剂量化疗药物可望克服肿瘤细胞中存在的化疗耐药和TRAIL耐受。     

Overcoming multi-drug resistance by anti-MDR1 ribozyme

AIM: To reverse multidrug resistance (MDR) of HepG2 by anti-MDR1 hammerhead ribozyme. METHODS: We developed an anti-MDR1 hammerhead ribozyme and delivered it to P-gp-overproducing human hepatocarcinoma cell line HepG2 by a retroviral vector containing RNA polymerase III promoter. We detected the expression of mdr1/Pgp and Rz in HepG2, HepG2 multidrug-resistant cell line and HepG2 Rz-transfected cells by real-time RT-PCR, semi-quantitative RT-PCR and Western blot methods. Moreover, MTT assay was tested to detect sensitivity of these ribozyme-transfected cells, and Rhodamine123 (Rh123) applied to test the function of Pgp. RESULTS: The Rz- transfected HepG2 cells became doxorubicin-sensitive, concomitant with the decreases in MDR1 expression, P-gp amounts and efflux pump function. CONCLUSION: The approaches using either retrovirus or liposome-mediated transfer of anti-MDR1 ribozyme may be selectively applicable to the treatment of MDR cells.     

Thyroid hormone export in rat FRTL-5 thyroid cells and mouse NIH-3T3 cells is carrier-mediated, verapamil-sensitive, and stereospecific.

Export of L-T3 out of the cell is one factor governing the cellular T3 content and response. We previously observed in liver-derived cells that T3 export was inhibited by verapamil, suggesting that it is due to either ATP-binding cassette/multidrug resistance (MDR1/mdr1b) or multidrug resistance-related (MRP1/mrp1) proteins. To test this hypothesis we measured T3 export in FRTL-5, NIH-3T3, and rat hepatoma (HTC) cells that varied in expression of these proteins. FRTL-5 and NIH-3T3 cells were found to contain a T3 efflux mechanism that is verapamil inhibitable, saturable, and stereospecific. By contrast, T3 efflux in HTC cells was slow and unaffected by verapamil. Neither FRTL-5 nor NIH-3T3 cells express mdrlb, but all three cell types express mrpl, as assessed by immunoblotting. Overexpression of MDR1 in NIH-3T3 cells did not enhance verapamil-inhibitable T3 efflux. Photoaffinity labeling of FRTL-5 and NIH-3T3 cells with [125I]L-T3 revealed a labeled 90- to 100-kDa protein that was not present in HTC cells. Verapamil and excess nonradioactive L-T3, but not D-T3, inhibited labeling of this protein. The lack of correlation between T3 efflux and MDR1 and mrpl expression and the finding of a photoaffinity-labeled putative transport protein smaller than MDR1 or mrp1 protein (approximately 170 kDa) suggest that a novel protein is involved in the transport of T3 out of cells.