JBC Avanti Polar Lipids

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M601266200 on May 3, 2006

J. Biol. Chem., Vol. 281, Issue 27, 18401-18407, July 7, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow An addition or correction has been published
Right arrow All Versions of this Article:
281/27/18401    most recent
M601266200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lee, T.-C.
Right arrow Articles by Huang, J.-d.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lee, T.-C.
Right arrow Articles by Huang, J.-d.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Enhanced Expression of Multidrug resistance-associated Protein 2 and Reduced Expression of Aquaglyceroporin 3 in an Arsenic-resistant Human Cell Line*

Te-Chang Lee{ddagger}1, I-Ching Ho{ddagger}, Wen-Jen Lu§, and Jin-ding Huang§

From the {ddagger}Institute of Biomedical Sciences, Academia Sinica, Taipei 11529 and the §Department of Pharmacology, National Cheng Kung University, Tainan 70101, Taiwan

Received for publication, February 9, 2006 , and in revised form, April 12, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Arsenic-resistant cells (R15), derived from a human lung adenocarcinoma cell line (CL3), were 10-fold more resistant to sodium arsenite (As(III)). Because R15 cells accumulated less arsenic than parental CL3 cells, this arsenic resistance may be due to higher efflux and/or lower uptake of As(III). We therefore compared expression of the multidrug resistance-associated proteins MRP1, MRP2, and MRP3 in these two cell lines. MRP2 expression was 5-fold higher in R15 cells than in CL3 cells, whereas MRP1 and MRP3 expression levels were similar. Furthermore, verapamil and cyclosporin A, inhibitors of multidrug resistance transporters, significantly reduced the efflux of arsenic from R15. Thus, increased arsenic extrusion by MRP2 may contribute to arsenic resistance in R15 cells. We also examined the expression of several aquaglyceroporins (AQPs), which mediate As(III) uptake by cells. Little AQP7 or AQP9 mRNA was detected by reverse transcription-PCR in either cell line, whereas AQP3 mRNA expression was 2-fold lower in R15 cells than in CL3 cells. When AQP3 expression in CL3 cells was knocked down by RNA interference, CL3 cells accumulated less arsenic and became more resistant to As(III). Conversely, overexpression of AQP3 in human embryonic kidney 293T cells increased arsenic accumulation, and the cells were more susceptible to As(III) than 293T cells transfected with vector alone. These results suggest that AQP3 is involved in As(III) accumulation. Taken together, our results suggest that enhanced expression of MRP2 and lower expression of AQP3 are responsible for lower arsenic accumulation in arsenic-resistant R15 cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Arsenic and its compounds are widely distributed in the environment and exhibit both metallic and nonmetallic properties (1, 2). Epidemiological studies and clinical observations show that chronic arsenic exposure increases the risk of developing vascular diseases and cancers of the skin, bladder, liver, kidney, and lung, as well as skin lesions in humans (3, 4). The International Agency for Research on Cancer categorizes arsenic and its compounds as known human carcinogens (5). Interestingly, As2O3 is a good chemotherapeutic agent for the treatment of acute promyelocytic leukemia (6, 7), and it shows promise for treatment of other cancers, including solid tumors (8). However, drug resistance, either intrinsic or acquired, limits the effectiveness of cancer treatments, thereby contributing to the death of some cancer patients receiving therapy. Thus, the possibility that patients may become resistant to arsenic treatment must be considered in advance of treatment (9, 10).

Acquired tolerance to arsenic in mammalian cells may be attributed to a variety of mechanisms, including elevated expression of GSH and glutathione S-transferase (11–14), elevated expression of heme oxygenase-1 (HO-1)2 (15), or increased efflux of arsenic via multidrug resistance-associated proteins (MRPs) or related transporters (12, 16–19). Because depletion of cellular GSH by treatment with buthionine sulfoximine reduces MRP-mediated efflux of arsenic and thus reverses the resistance to arsenic in multidrug-resistant cell lines (20), MRP family transporters have been proposed to play a crucial role in extruding intracellular arsenic and/or As(III)-GSH complexes (17, 19, 21, 22).

In contrast to studies on arsenic efflux, there have been few reports related to arsenic uptake by eukaryotic cells. Pentavalent arsenate (As(V)), the predominant form of arsenic in nature, can be taken up by cells through phosphate transporters (23). Recently, aquaglyceroporins (AQPs) 7 and 9 were shown to transport As(III) into mammalian cells (24–26). Homologous transporter systems responsible for the uptake of As(III) and antimony have been identified in a variety of organisms; for example, GlpF protein in Escherichia coli (27), FPS1 protein in Saccharomyces cerevisiae (28), and LmAQP1 protein in Leishmania major and Leishmania arentolae (29). The leukemia cell line K562, when transfected with the AQP9 gene, became hypersensitive to As2O3 because of an increased rate of As(III) uptake (30). Furthermore, GSH conjugates of As(III) can be taken up by cells via organic anion transporting polypeptide C (OATP-C) (31), and hexose permeases reportedly facilitate As(III) uptake in S. cerevisiae (32).

We previously established the arsenic-resistant cell lines SA7 and CL3R15 (abbreviated R15) from Chinese hamster ovary cells (11) and human lung adenocarcinoma (CL3) cells (15), respectively. SA7 cells exhibit higher levels of GSH and glutathione S-transferase {pi} than parental Chinese hamster ovary cells (11, 33). Our studies have shown that both GSH and glutathione S-transferase {pi} are involved in the extrusion of arsenic from cells (16, 34). In addition, arsenic extrusion in SA7 cells is ATP-dependent and inhibited by verapamil (VP) and cyclosporin A (CSA), two inhibitors of multidrug resistance transporters (16). We have also demonstrated the constitutive expression of HO-1 in R15 cells (15). HO-1 participates in a protective mechanism against oxidative stress in various tissues (35), and our results show that HO-1 also contributes to the protection of cells from arsenic cytotoxicity (15). However, arsenic tolerance is mainly due to increased arsenic efflux and/or decreased arsenic uptake, resulting in reduced cellular arsenic burden. To further explore the mechanisms of arsenic tolerance in R15 cells, we compared the expression of several transporter genes (AQP3, AQP7, AQP9, MRP1, MRP2, and MRP3) in CL3 and R15 cells and investigated their involvement in arsenic accumulation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—As(III), trivalent sodium arsenite, was obtained from Merck (Darmstadt, Germany). The media and chemicals used for cell culture were purchased from Invitrogen. Fetal bovine serum was obtained from HyClone Laboratories, Inc. (Logan, UT). VP was purchased from Sigma, and CSA was from Novartis (Basel, Switzerland). Antibodies against AQP3, {alpha}-tubulin, beta-actin, MRP1 (QCRL-3), MRP2 (H-300), and MRP3 (C-18) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Cell Lines—All cells were grown in media supplemented with 10% heat-inactivated fetal bovine serum at 37 °C in a humidified atmosphere with 5% CO2. CL3, a human lung adenocarcinoma cell line, was grown in F12 medium (15). Arsenic-resistant R15 cells were maintained in F12 medium containing 4 µM As(III). HepG2 cells were grown in Earle's minimum essential medium, and human embryonic kidney 293T cells were cultured in Dulbecco's modified Eagle's medium.

Cytotoxicity Assay—The survival rate of cells after drug treatment was determined with a colony-forming assay (36). In brief, 2–3 x 105 CL3 or R15 cells were plated on a 35-mm dish 1 day prior to chemical treatment. The cultures were treated with various concentrations of tested compounds for 6 h. At the end of treatment, the cells were replated at 150 cells per 60-mm dish, in triplicate, and incubated in fresh medium supplemented with 0.1 µg/ml insulin and 5 µM hydrocortisone for 9 days. The medium was refreshed once at day 5. The colonies (>50 cells) were fixed, stained, and counted as described previously (36). Under certain circumstances, the cell viability immediate after treatment was determined by dye exclusion assay. In brief, the cell suspension was mixed with an equal volume of trypan blue stain solution (Invitrogen), and the viable cells were counted under a microscope using a hemocytometer.

Arsenic Determination—Cellular levels of arsenic were determined by atomic absorption spectrophotometry (AAS, Hitachi Z-8000, Tokyo) as described previously (36). In brief, cells were plated on a 100-mm dish and treated with As(III) using protocols described in the figure legends. After treatment, the cells were washed five times with phosphate-buffered saline containing 1 mM EDTA, trypsinized, digested with nitric acid, and subjected to arsenic determination by an atomic absorption spectrophotometer equipped with a hydride formation system.

Real-time Quantitative PCR Analysis of MRP mRNA—Total cellular RNA was extracted from CL3 and R15 cells using an RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. MRP1, MRP2, and MRP3 mRNA levels were determined by real-time quantitative PCR on the LightCycler (Roche Diagnostics, Mannheim, Germany) using TaqMan probes. Real-time PCR was performed in glass capillaries with an initial denaturation step of 10 min at 95 °C followed by 50 cycles of 95 °C for 10 s and 60 °C for 30 s in a total volume of 10 µl containing 1 µl of 10x reaction buffer, 150 nM TaqMan probe, 500 nM each primer, 3 mM MgCl2, and 2.5 µl of reverse transcription product. The mRNA levels of MRP1, MRP2, and MRP3 were quantified using LightCycler analysis software, version 3.5, and expressed as concentrations relative to GAPDH mRNA. The sequences of the primer pairs and TaqMan probes for MRP1, MRP2, and MRP3 are summarized in Table 1.


View this table:
[in this window]
[in a new window]
  		TABLE 1
Summary of primer sequences

 
One-step RNA PCR Analysis of AQP mRNAs—Total cellular RNA was extracted using TRI reagent (Molecular Research Center, Cincinnati, OH). AQP3, AQP7, and AQP9 mRNA levels were estimated using the GeneAmp Gold RNA PCR reagent kit (Applied Biosystems, Foster City, CA). The amplified products were visualized on a 2% agarose gel. The primers for AQP3, AQP7 and AQP9, listed in Table 1, were prepared according to those reported in Liu et al. (26). GAPDH mRNA was used as a loading control.

Western Blot Analysis—Expression of MRP1, MRP2, MRP3, and AQP3 proteins was determined by Western blotting (37). In brief, the proteins in an aliquot of cell lysate (10–25 µg of protein) were electrophoretically separated on a sodium dodecyl sulfate-polyacrylamide gel (7 or 10% acrylamide) and then transferred onto a polyvinylidene difluoride membrane using a semidry electrotransfer system (ATTO, Tokyo Japan). The membrane was blocked in 5% bovine serum albumin in TBS-T (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, and 0.1% (v/v) Tween 20) for 1 h at room temperature and then incubated with primary antibody in 2.5% bovine serum albumin in TBS-T overnight at 4 °C. After five washes with TBS-T, the membrane was incubated with secondary antibody for 60 min followed by another five washes with TBS-T. Immunopositive bands were detected using ECL Western blotting detection reagents (Amersham Biosciences). {alpha}-Tubulin or beta-actin was used as an internal control. Protein concentrations were determined by Bradford analysis using bovine serum albumin as the standard (38).

Knock Down of AQP3 by RNA Interference—The AQP3 mRNA nucleotide sequence (NM_004925 [GenBank] ) from position 1758 to 1776 (5'-TCCTCTACTCAACAATGTA-3') was used to construct an expression vector to generate small interfering RNA (siRNA) (pAQP3-siRNA). We synthesized a double-stranded oligonucleotide 5'-TCGAC(X19)TTCG(Y19)TTTT-3' and inserted it into the SalI and XbaI sites of a p3in1 vector (a gift from Dr. W. Y. Hu, The Salk Institute, La Jolla, CA), where X19 is the selected sequence and Y19 is its reversed/complementary sequence. The pAQP3-siRNA and p3in1 vector were then transfected into CL3 cells using FuGENE 6 (Roche Diagnostics). In brief, 2 x 105 cells were plated in a 6-well plate 1 day prior to transfection. Plasmid DNA (1 µg) in FuGENE 6 reagent (3 µl) was preincubated for 15 min at room temperature and then added to cell cultures that were then incubated for 48 h at 37 °C. Cells resistant to G418 (Geneticin, Invitrogen) were selected by incubation in the presence of 200 µg/ml G418 for 3 weeks. The accumulation of As(III) and sensitivity of CL3 cells (transfected with AQP3 siRNA or vector alone) to As(III) for 6 h was determined using AAS and the colony-forming assay, respectively.

Overexpression of AQP3 in 293T Cells—pFLAG-CMV22-AQP3 was constructed by inserting the reverse transcription-PCR-amplified full-length human AQP3 cDNA (sequence verified) into the EcoRI site of pFLAG-CMV22 (obtained from Sigma). pFLAG-CMV22-AQP3 and pFLAG-CMV22 were transfected into human embryonic kidney 293T cells using FuGENE 6, as described above. After a 48-h incubation, the AQP3 expression level was determined by Western blotting using antibodies against either FLAG or AQP3. After the transfected cells were incubated with As(III) for 10 min, the initial As(III) uptake was determined using AAS. The relative survival was analyzed by the colonyforming assay after exposure to various concentrations of As(III) for 6 h.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Increased Arsenic Extrusion in Arsenic-resistant R15 Cells—Arsenic-resistant R15 cells were derived by growing CL3 cells in medium while progressively increasing the As(III) concentration to 4 µM (15). The resistance of R15 cells was confirmed by a colony-forming assay (Fig. 1). Using a 6-h exposure, the IC50 values were estimated to be 10 and 100 µM for CL3 and R15 cells, respectively. To understand whether altered arsenic transport was involved in arsenic resistance in R15 cells, we used AAS to determine the intracellular accumulation of arsenic in CL3 and R15 cells. We first assessed the viability of the cells under the experimental uptake conditions. Immediately after treatment of CL3 and R15 cells with 20 µM As(III) for up to 8 h, the cell viability examined by a dye exclusion assay was 88.3 ± 4.5% and 97.7 ± 6.7%, respectively. The rate of arsenic accumulation in R15 cells was much slower than in CL3 cells after incubation with 20 µM As(III) for up to 8 h (Fig. 2A). In the parental CL3 cells, arsenic accumulation increased throughout the 8-h incubation period; in fact, arsenic accumulation did not reach a steady state even after 24 h (data not shown). In contrast, arsenic accumulation in R15 cells reached a steady state after 60 min and did not increase significantly thereafter (Fig. 2A). Next, we compared the rate of arsenic extrusion in CL3 and R15 cells after treatment with 100 µM As(III) for 30 min. As shown in Fig. 2B, the half-life of arsenic elimination in R15 cells was ~15 min, whereas the half-life in CL3 cells was ~45 min. These kinetic data suggest that the activity of an efflux transporter was enhanced in R15 cells. The dye exclusion assay confirmed that the cells maintained their integrity (>95%) after treatment with 100 µM As(III) for 30 min.

Overexpression of MRP2 in Arsenic-resistant R15 Cells—We evaluated the mRNA and protein levels of efflux transporters in CL3 and R15 cells using real-time quantitative PCR and Western blotting. Fig. 3A shows that the relative concentration of MRP2 mRNA was 5.1 ± 0.4-fold higher in R15 cells than in CL3 cells, whereas the relative MRP1 and MRP3 mRNA levels were similar, with ratios of 1.3 ± 0.1 and 1.0 ± 0.2, respectively. Western blot analysis confirmed the higher level of MRP2 protein in R15 cells as compared with CL3 cells (Fig. 3B). There was no significant difference in MRP1 and MRP3 protein levels between cell lines, consistent with their relative mRNA levels analyzed by real-time PCR. When the vinblastine-resistant leukemia cell line CEMR and its parental counterpart CEM were used as the respective negative and positive controls, little P-glycoprotein (MDR1) expression was detected in either CL3 or R15 cells by either reverse transcription and PCR amplification or Western blotting (data not shown). To investigate the possible correlation between MRP2 overexpression and arsenic resistance in R15 cells, we treated CL3 and R15 cells with As(III) in the presence or absence of the MRP inhibitors VP and CSA and subsequently examined arsenic accumulation and cellular sensitivity. The addition of either VP or CSA significantly increased the sensitivity of R15, but not CL3 cells, to As(III) (Fig. 4, A and B). Furthermore, VP and CSA each increased arsenic accumulation in R15 cells in a dose-dependent manner but did not change the arsenic content of CL3 cells (Fig. 4, C and D). Similar to other reports (22, 39), our results suggest that MRP2 has a crucial role in eliminating arsenic from R15 cells.


Figure 1
View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 1.
Sensitivity of CL3 and R15 cells to As(III). Logarithmically growing CL3 and R15 cells were treated with various concentrations of As(III) for 6 h. Relative survival was analyzed by a colony-forming assay as described under "Experimental Procedures." The average colony-forming efficiencies for untreated CL3 and R15 cells were 73.3 ± 13.4 and 56.4 ± 12.0, respectively. Bars, S.D. of three independent experiments.

 
Lower Arsenic Uptake Rate and Lower Expression Levels of AQP3 in R15 Cells—The lower arsenic accumulation in R15 cells during the first 60 min (Fig. 2A) may additionally imply the down-regulation of an influx transporter. We therefore examined the expression of several influx channels or transporters, AQP3, AQP7, and AQP9, in CL3 and R15 cells. Neither AQP7 nor AQP9 mRNA expression was detected in either CL3 or R15 cells by GeneAmp Gold RNA PCR (Fig. 5A). However, the AQP3 mRNA expression level in R15 cells was approximately half that of CL3 cells (Fig. 5B). Western blot analysis confirmed the lower protein levels of AQP3 in R15 cells (Fig. 5B).

To validate the importance of AQP3 in As(III) uptake, RNA interference was used to suppress AQP3 gene expression (40). Transfection of the AQP3 siRNA expression vector into CL3 cells decreased AQP3 expression by approximately half (Fig. 6A) and decreased both arsenic accumulation (Fig. 6B) and sensitivity to As(III) (Fig. 6C). The IC50 values for a 6-h exposure were 9.7 ± 2.9 and 20.3 ± 3.2 µM for CL3 transfected with control vector and AQP3 siRNA, respectively (p < 0.001 by Student's t test). Furthermore, transfection of an expression vector encoding FLAG-tagged AQP3 into human embryonic kidney 293T cells increased expression of AQP3 (Fig. 7A) and enhanced the initial arsenic uptake following a 10-min incubation with As(III) (Fig. 7B). We also observed decreased As(III) resistance in AQP3-transfected 293T cells (Fig. 7C). The IC50 values for a 6-h exposure were 20.0 ± 3.2 and 10.0 ± 0.9 µM for 293T cells transfected with control vector and FLAG-AQP3, respectively (p < 0.001 by Student's t test). These results imply that AQP3 is involved in As(III) uptake in CL3 cells. Real-time quantitative PCR and Western blotting revealed no difference in the expression of OATP-C, an arsenic transporter that mediates the uptake of GSH conjugates of arsenite, in CL3 and R15 cells (31) (data not shown).


Figure 2
View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 2.
Arsenic (As) accumulation and extrusion in CL3 and R15 cells. A, logarithmically growing CL3 and R15 cells were treated with 20 µM As(III) for 1–8 h. At the times indicated, cells were harvested by trypsinization, and intracellular arsenic content was determined by AAS as described under "Experimental Procedures." B, CL3 and R15 cells were first fed with 100 µM As(III) for 30 min, washed twice with PBS, and then incubated in drug-free medium for 15–120 min. The amount of arsenic remaining in the cells was determined by AAS. Bars, S.D. of three independent experiments.

 


Figure 3
View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 3.
Comparison of relative MRP1, MRP2, and MRP3 mRNA and protein levels in CL3 and R15 cells. A, real-time quantitative PCR analysis of MRP1, MRP2, and MRP3 mRNA levels. Total cellular RNA from CL3 and R15 cells was extracted, and the MRP1, MRP2, and MRP3 mRNA levels were determined relative to GAPDH expression by real-time quantitative PCR using TaqMan probes. Data are the means ± S.D. (n = 6). ***, p < 0.001. B, Western blot analysis of MRP1, MRP2, and MRP3. Logarithmically growing CL3 and R15 cells were lysed, and the proteins were separated by electrophoresis on a 7% SDS-polyacrylamide gel. MRP1, MRP2, and MRP3 protein levels were detected by Western blotting. {alpha}-Tubulin was used as a loading control.

 


Figure 4
View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 4.
Effect of VP and CSA on arsenic (As) accumulation and cytotoxicity in CL3 and R15 cells. A and B, CL3 and R15 cells were exposed to increasing concentrations of As(III) in the absence or presence of 50 µM VP (A) or 5 µg/ml CSA (B) for 6 h. After incubation, relative cell survival was determined by the colony-forming assay. C and D, CL3 and R15 cells were treated with 10 µM As(III) and 100 µM As(III), respectively, and various concentrations of VP (C) or CSA (D) for 6 h. After treatment, intracellular arsenic accumulation was determined by AAS. Bars, S.D. of three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Arsenic-resistant R15 cells, derived from the human lung adenocarcinoma cell line CL3, have several distinct characteristics (15). We previously demonstrated that HO-1 is up-regulated and cyclooxygenase-2 (COX-2) is down-regulated in R15 cells (15, 41). The lower level of COX-2 renders R15 cells more resistant to benzo(a)pyrene (41). The mechanism of arsenic resistance in R15 cells, however, remained unexplained despite the fact that these cells have been established for some time. However, recent advances in our knowledge of arsenic transporters and channels enabled us to examine the mechanisms of arsenic resistance in this regard. By examining the kinetics of arsenic accumulation in CL3 and R15 cells, we demonstrated that the amount of arsenic accumulation during the first 60 min was apparently lower in R15 cells than in the parental CL3 cells (Fig. 2A). Furthermore, in R15 cells, the accumulated arsenic had a short half-life and quickly reached a lower steady state than in the parental CL3 cells, in which arsenic accumulation did not plateau for at least 24 h. These kinetic data imply that R15 cells exhibit fast extrusion and/or slow uptake of arsenic.


Figure 5
View larger version (72K):
[in this window]
[in a new window]
  		FIGURE 5.
Expression of AQP3, AQP7, and AQP9 in CL3 and R15 cells. A, total cellular RNA was extracted, and AQP3, AQP7, and AQP9 mRNA levels were determined using the TaqMan one-step RNA PCR reagent kit. Lane M, marker; lane A, CL3 cells; lane B, R15 cells; lane C, HepG2 cells. HepG2 cells, a human hepatoma cell line, were used as a positive control for AQP7 and AQP9 expression. GAPDH mRNA was used as a loading control. B, Western blot analysis of AQP3 in CL3 and R15 cells. Logarithmically growing CL3 and R15 cells were lysed and separated by electrophoresis on a 10% SDS-polyacrylamide gel. AQP3 protein was detected by Western blotting. The relative levels of the proteins were determined by quantitation of the immunopositive bands with a densitometer.

 
Our observation that the half-life for arsenic elimination in R15 and CL3 cells was 15 and 45 min, respectively (Fig. 2B), suggests that R15 cells extrude arsenic more efficiently than CL3 cells. We examined the expression levels of several possible arsenic efflux transporters, and our results showed that CL3 and R15 cells did not differ in their expression levels of MRP1 and MRP3; the level of MRP2, however, was 5-fold higher in R15 cells. As(III) up-regulates MRP2 gene expression via activation of the c-Jun NH2-terminal kinase (JNK) pathway in primary rat and human hepatocytes (39). It is not clear, however, why As(III) specifically enhanced the expression of MRP2, but not MRP1 and MRP3, in R15 cells. The enhanced arsenic efflux in R15 cells is therefore likely due to the higher expression of MRP2, which pumps arsenite triglutathionate out of cells (22, 39). Arsenite triglutathionate is generally transported by MRP1 and MRP2 (19). As reported previously, R15 cells have higher levels of GSH than CL3 cells (15). VP and CSA, usually used to inhibit multidrug resistance transporters including P-glycoproteins and MRPs (42, 43), significantly inhibited arsenic extrusion and thus decreased arsenic resistance in R15 cells (Fig. 3). These results are consistent with our previous study showing that VP and CSA inhibit arsenic efflux in arsenic-resistant Chinese hamster ovary cells (16). The inhibitory effects of VP and CSA on arsenic efflux support the idea that MRP2-mediated arsenic extrusion is one of the important mechanisms of arsenic resistance in R15 cells.


Figure 6
View larger version (26K):
[in this window]
[in a new window]
  		FIGURE 6.
Reduced sensitivity of CL3 cells to As(III) upon knockdown of AQP3 expression using RNA interference. CL3 cells were transfected with either pAQP3-siRNA or the control p3in1 vector using the FuGENE 6 reagent, and G418-resistant cells were selected with 200 µg/ml G418 for 3 weeks. A, the AQP3 protein level was determined by Western blotting. beta-Actin was used as a loading control. The relative levels of the proteins were determined by quantitation of the immunopositive bands with a densitometer. p3in1 control vector- and pAQP3-siRNA-transfected CL3 cells treated with As(III) for 6 h were subjected to AAS for the determination of arsenic (As) accumulation (B) and As(III) sensitivity by the colony-forming assay (C). The average colony-forming efficiencies for CL3 cells transfected with pAQP3-siRNA and p3in1 were 41.0 ± 6.5 and 46.0 ± 4.9, respectively. Bars, S.D. of three independent experiments. *, p < 0.05 by Student's t test.

 
Similarly, P-glycoprotein and MRP1 levels have been found to be elevated in As(III)-treated cells (44). Numerous reports have shown an association between P-glycoprotein and MRP1 expression and arsenic toxicity (20, 45–47). In contrast, no cross-resistance to As2O3 has been observed in human leukemia MOLT-4 cells (48) or HL60 cells (49) overexpressing P-glycoprotein. Takeshita et al. (44) demonstrated that arsenic-resistant NB4 cells, an acute promyelocytic leukemia cell line, express higher levels of P-glycoprotein and MRP1 than the parental cells but have similar levels of arsenic accumulation. We found little P-glycoprotein in either CL3 or R15 cells and no difference in the amount of MRP1 (Fig. 3). The increased susceptibility of R15 cells to VP and CSA is apparently not due to their inhibitory effects on P-glycoprotein or MRP1. However, we cannot rule out an indirect mechanism, for example, that an inhibitor-mediated decrease in GSH efflux via MRP1 may increase cellular GSH levels and thus facilitate efflux of arsenic-GSH complexes via MRP2. This putative mechanism requires further investigation. Participation of a cell type-dependent transporter for arsenic efflux is another hypothesis worthy of consideration.


Figure 7
View larger version (26K):
[in this window]
[in a new window]
  		FIGURE 7.
Increased sensitivity of 293T cells to As(III) by overexpression of FLAG-tagged AQP3. pFLAG-CMV22-AQP3 or pFLAG-CMV22 (vector control) was transfected into 293T cells using the FuGENE 6 reagent. A, the expression of AQP3 was determined by Western blotting using primary antibodies against FLAG or AQP3. B, the transfected cells were exposed to various concentrations of As(III) for 10 min. Initial arsenic (As) uptake was determined by AAS. *, **, and *** are p < 0.05, 0.01, and 0.001, respectively, (Student's t test) as compared with cells transfected with control vector. C, pFLAG-CMV22-AQP3- and pFLAG-CMV22-transfected cells were exposed to various concentrations of As(III) for 6 h, and their relative survival was analyzed by the colony-forming assay. The average colony-forming efficiencies for 293T cells transfected with pFLAG-CMV22-AQP3 and pFLAG-CMV22 were 45.3 ± 17.6 and 48.3 ± 10.2, respectively. Bars, S.D. of three independent experiments.

 
Down-regulation of an uptake system is another mechanism that may yield acquired resistance. Recent evidence has shown that As(OH)3, an aqueous form of As(III) (25), is taken into cells via AQP pathways (24, 26–28). By expressing various AQP genes in yeast or Xenopus oocytes (24, 26), AQP9 and AQP7 were shown to be efficient channels for As(OH)3, whereas there was little or no transport by hAQP3 or hAQP10 (26). Transfection of the L. major homologue of mammalian AQP9, LmAQP1, into three species of Leishmania produced hypersensitivity to both As(III) and potassium antimonyl tartrate in all three strains (29), suggesting a strategy for drug resistance reversal. Unfortunately, we found no AQP7 or AQP9 expression in CL3 and R15 cells, whereas the AQP3 expression level in R15 cells was reduced to half that of CL3 cells (Fig. 5). Although previous studies have shown that AQP3 has limited capacity to transport arsenic (24, 26), our present study using RNA interference (Fig. 6) and ectopic expression (Fig. 7) verifies that AQP3 participates in As(III) uptake in lung adenocarcinoma cells.

AQPs are channels for water, glycerol, and other neutral solutes (50), and their discovery has provided new insights into the molecular basis of membrane permeability (51). Recent evidence also shows that AQPs are involved in cell migration, skin hydration, and fat metabolism (52). However, AQP expression is tissue-dependent in mammals. AQP9 channels, highly expressed in liver and leukocytes (53, 54), are the likely route of As2O3 uptake into leukemia cells. AQP7, mainly present in adipocytes, functions as a channel for glycerol release during fasting (55). In the fasting state, the expression of both AQP7 and AQP9 increases to allow glycerol release from fat cells and glycerol uptake into hepatocytes, respectively (56). AQP3 is widely expressed in epithelial tissues of the urinary, digestive, respiratory, and integumentary systems (57). In AQP3-null mice, stratum corneum hydration and skin elasticity are reduced (58). Accumulated evidence has shown that the eukaryotic AQPs are often regulated by phosphorylation, pH, osmolarity, and binding of other proteins or ligands (59, 60). Our current study, as well as many others (30), implies that AQPs could be potential drug targets to enhance the efficacy of chemotherapeutic agents. Human OATP-C transporter was recently reported to be an influx transporter for As(III) in the liver (31). However, we found no difference in OATP-C expression between R15 and CL3 cells (data not shown).

Resistance to chemotherapy, a major problem in the management of cancer, is the result of the interplay among uptake, efflux, metabolic inactivation, and sequestration of the drug. All organisms from bacteria to mammals have similar systems for arsenic detoxification, including As(V) uptake via phosphate transporters and As(III) uptake by AQPs, reduction of As(V) to As(III) by a reductase, and extrusion or sequestration of As(III) by MRPs (25). In this study, we observed a 10-fold increase in the IC50 in R15 cells as compared with CL3 cells. This change can be explained by a 2-fold decrease in uptake and a 5-fold increase in elimination. This study does not rule out mechanisms other than the two we have addressed. Nevertheless, our results suggest that the function of AQP3 and MRP2, important factors that regulate arsenic levels in cells, could be modulated to enhance the efficacy of the arsenic trioxide regimen during cancer treatment.


   FOOTNOTES
 
* This study was supported by Grant AS91IBC3PP from the Academia Sinica, Grants NSC 92-2320-B-010-017, NSC93-2320-B-001-053, and NSC92-3112-B006-016 from the National Science Council, and Grant NHRI-EX95-9522BI from the National Health Research Institutes (to T.-C. L.) and Grant NSC93-3112-B006-005 from the National Science Council of the Republic of China (Taipei, Taiwan) (to J.-d. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. Tel.: 886-2-26523055; Fax: 886-2-27829142; E-mail: bmtcl{at}ibms.sinica.edu.tw.

2 The abbreviations used are: HO-1, heme oxygenase-1; MRP, multidrug resistance-associated protein; AQP, aquaglyceroporin; VP, verapamil; CSA, cyclosporin A; AAS, atomic absorption spectrophotometry; siRNA, small interfering RNA; OATP-C, organic anion transporting polypeptide C; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Ling-Huei Yih and Jeng-Fan Lo for their critical review of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Nordstrom, D. K. (2002) Science 296, 2143–2145[CrossRef][Medline] [Order article via Infotrieve]
  2. Smith, A. H., Lopipero, P. A., Bates, M. N., and Steinmaus, C. M. (2002) Science 296, 2145–2146[Abstract/Free Full Text]
  3. Pinto, S. S., and Nelson, K. W. (1976) Annu. Rev. Pharmacol. Toxicol. 16, 95–100[Medline] [Order article via Infotrieve]
  4. Chen, C. J., Chuang, Y. C., Lin, T. M., and Wu, H. Y. (1985) Cancer Res. 45, 5895–5899[Medline] [Order article via Infotrieve]
  5. International Agency for Research on Cancer (IARC) (1987) in IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans (WHO IARC, ed) Vol. 23, pp. 39–142, IARC, Lyon, France
  6. Soignet, S. L., Maslak, P., Wang, Z.-G., Jhanwar, S., Calleja, E., Dardashti, L. J., Corso, D., Deblasio, A., Gabrilove, J., Scheinberg, D. A., Pandolfi, P. P., and Warrell, R. (1998) N. Engl. J. Med. 339, 1341–1348[Abstract/Free Full Text]
  7. Niu, C., Yan, H., Yu, T., Sun, H.-P., Liu, J.-X., Li, X.-S., Wu, W., Zhang, F.-Q., Chen, Y., Zhou, L., Li, J.-M., Zeng, X.-Y., Ou Yang, R.-R., Yuan, M.-M., Ren, M.-Y., Gu, F.-Y., Cao, Q., Gu, B.-W., Su, X.-Y., Chen, G.-Q., Xiong, S.-M., Zhang, T.-D., Waxman, S., Wang, Z.-Y., Chen, Z., Hu, J., Shen, Z.-X., and Chen, S.-J. (1999) Blood 94, 3315–3324[Abstract/Free Full Text]
  8. Evens, A. M., Tallman, M. S., and Gartenhaus, R. B. (2004) Leuk. Res. 28, 891–900[CrossRef][Medline] [Order article via Infotrieve]
  9. Kerbauy, D. M., Lesnikov, V., Abbasi, N., Seal, S., Scott, B., and Deeg, H. J. (2005) Blood 106, 3917–3925[Abstract/Free Full Text]
  10. Takeshita, A., Shinjo, K., Naito, K., Matsui, H., Sahara, N., Shigeno, K., Horii, T., Shirai, N., Maekawa, M., Ohnishi, K., Naoe, T., and Ohno, R. (2005) Leukemia (Basingstoke) 19, 1306–1311
  11. Lee, T.-C., Wei, M.-L., Chang, W.-J., Ho, I.-C., Jan, K. Y., and Huang, H. (1989) In Vitro Cell. Dev. Biol. 25, 442–448[Medline] [Order article via Infotrieve]
  12. Liu, J., Chen, H., Miller, D. S., Saavedra, J. E., Keefer, L. K., Johnson, D. R., Klaassen, C. D., and Waalkes, M. P. (2001) Mol. Pharmacol. 60, 302–309[Abstract/Free Full Text]
  13. Brambila, E. M., Achanzar, W., Qu, W., Webber, M. M., and Waalkes, M. P. (2002) Toxicol. Appl. Pharmacol. 183, 99–107[CrossRef][Medline] [Order article via Infotrieve]
  14. Davison, K., Côté, S., Mader, S., and Miller, W. H. (2003) Leukemia (Basingstoke) 17, 931–940
  15. Lee, T. C., and Ho, I. C. (1994) Cancer Res. 54, 1660–1664[Abstract/Free Full Text]
  16. Huang, R. N., and Lee, T. C. (1996) Toxicol. Appl. Pharmacol. 141, 17–22[Medline] [Order article via Infotrieve]
  17. Zaman, G. J. R., Lankelma, J., van Tellingen, O., Beijnen, J., Dekker, H., Paulusma, C., Oude Elferink, R. P. J., Baas, F., and Borst, P. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7690–7694[Abstract/Free Full Text]
  18. Vernhet, L., Allain, N., Bardiau, C., Anger, J. P., and Fardel, O. (2000) Toxicology 142, 127–134[Medline] [Order article via Infotrieve]
  19. Leslie, E. M., Haimeur, A., and Waalkes, M. P. (2004) J. Biol. Chem. 279, 32700–32708[Abstract/Free Full Text]
  20. Vernhet, L., Allain, N., Payen, L., Anger, J.-P., Guillouzo, A., and Fardel, O. (2001) Biochem. Pharmacol. 61, 1387–1391[CrossRef][Medline] [Order article via Infotrieve]
  21. Keppler, D. (1999) Free Rad. Biol. Med. 27, 985–991[CrossRef][Medline] [Order article via Infotrieve]
  22. Kala, S. V., Neely, M. W., Kala, G., Prater, C. I., Atwood, D. W., Rice, J. S., and Lieberman, M. W. (2000) J. Biol. Chem. 275, 33404–33408[Abstract/Free Full Text]
  23. Huang, R.-N., and Lee, T.-C. (1996) Toxicol. Appl. Pharmacol. 136, 243–249[CrossRef][Medline] [Order article via Infotrieve]
  24. Liu, Z., Shen, J., Carbrey, J. M., Mukhopadhyay, R., Agre, P., and Rosen, B. P. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 6053–6058[Abstract/Free Full Text]
  25. Rosen, B. P. (2002) FEBS Lett. 529, 86–92[CrossRef][Medline] [Order article via Infotrieve]
  26. Liu, Z., Carbrey, J. M., Agre, P., and Rosen, B. P. (2004) Biochem. Biophys. Res. Commun. 316, 1178–1185[CrossRef][Medline] [Order article via Infotrieve]
  27. Sanders, O. I., Rensing, C., Kuroda, M., Mitra, B., and Rosen, B. P. (1997) J. Bacteriol. 179, 3365–3367[Abstract/Free Full Text]
  28. Wysocki, R., Chéry, C. C., Wawrzycka, D., Van Hulle, M., Cornelis, R., Thevelein, J. M., and Tamás, M. J. (2001) Mol. Microbiol. 40, 1391–1401[CrossRef][Medline] [Order article via Infotrieve]
  29. Gourbal, B., Sonuc, N., Bhattacharjee, H., Legare, D., Sundar, S., Ouellette, M., Rosen, B. P., and Mukhopadhyay, R. (2004) J. Biol. Chem. 279, 31010–31017[Abstract/Free Full Text]
  30. Bhattacharjee, H., Carbrey, J. M., Rosen, B. P., and Mukhopadhyay, R. (2004) Biochem. Biophys. Res. Commun. 322, 836–841[CrossRef][Medline] [Order article via Infotrieve]
  31. Lu, W.-J., Tamai, I., Nezu, J.-I., Lai, M.-L., and Huang, J.-D. (2006) J. Biomed. Sci., in press
  32. Liu, Z., Boles, E., and Rosen, B. P. (2004) J. Biol. Chem. 279, 17312–17318[Abstract/Free Full Text]
  33. Lo, J. F., Wang, H. F., Tam, M. F., and Lee, T. C. (1992) Biochem. J. 288, 977–981[Medline] [Order article via Infotrieve]
  34. Wang, H. F., and Lee, T. C. (1993) Biochem. Biophys. Res. Commun. 192, 1093–1099[CrossRef][Medline] [Order article via Infotrieve]
  35. Dennery, P. A., Sridhar, K. J., Lee, C. S., Wong, H. E., Shokoohi, V., Rodgers, P. A., and Spitz, D. R. (1997) J. Biol. Chem. 272, 14937–14942[Abstract/Free Full Text]
  36. Ho, I.-C., and Lee, T.-C. (1999) Toxicol. Sci. 47, 176–180[Abstract/Free Full Text]
  37. Burnette, W. H. (1981) Anal. Biochem. 112, 195–203[CrossRef][Medline] [Order article via Infotrieve]
  38. Bradford, M. M. (1976) Anal. Biochem. 72, 248–254[CrossRef][Medline] [Order article via Infotrieve]
  39. Vernhet, L., Seite, M. P., Allain, N., Guillouzo, A., and Fardel, O. (2001) J. Pharamcol. Exp. Ther. 298, 234–239[Abstract/Free Full Text]
  40. Reynolds, A., Leake, D., Boese, Q., Scaringe, S., Marshall, W. S., and Khvorova, A. (2004) Nat. Biotechnol. 22, 326–330[CrossRef][Medline] [Order article via Infotrieve]
  41. Ho, I. C., and Lee, T. C. (2002) J. Toxicol. Environ. Health Part A 65, 245–263[CrossRef][Medline] [Order article via Infotrieve]
  42. Cole, S. P., Bhardwaj, G., Gerlach, J. H., Mackie, J. E., Grant, C. E., Almquist, K. C., Stewart, A. J., Kurz, E. U., Duncan, A. M., and Deeley, R. G. (1992) Science 258, 1650–1654[Abstract/Free Full Text]
  43. Juliano, R. L., and Ling, V. (1976) Biochim. Biophys. Acta 455, 152–162[Medline] [Order article via Infotrieve]
  44. Takeshita, A., Shinjo, K., Naito, K., Matsui, H., Shigeno, K., Nakamura, S., Horii, T., Maekawa, M., Kitamura, K., Naoe, T., Ohnishi, K., and Ohno, R. (2003) Leukemia (Basingstoke) 17, 650–651
  45. Cole, S. P. C., Sparks, K. E., Fraser, K., Loe, D. W., Grant, C. E., Wilson, G. M., and Deeley, R. G. (1994) Cancer Res. 54, 5902–5910[Abstract/Free Full Text]
  46. Liu, J., Liu, Y., Powell, D. A., Waalkes, M. P., and Klaassen, C. D. (2002) Toxicology 170, 55–62[CrossRef][Medline] [Order article via Infotrieve]
  47. Kimura, A., Ishida, Y., Wada, T., Yokoyama, H., Mukaida, N., and Kondo, T. (2005) Toxicol. Appl. Pharmacol. 203, 53–61[CrossRef][Medline] [Order article via Infotrieve]
  48. Hu, X. M., Hirano, T., and Oka, K. (2003) Cancer Chemother. Pharmacol. 51, 119–126[CrossRef][Medline] [Order article via Infotrieve]
  49. Perkins, C., Kim, C. N., Fang, G., and Bhalla, K. N. (2000) Blood 95, 1014–1022
  50. Agre, P., and Kozono, D. (2003) FEBS Lett. 555, 72–78[CrossRef][Medline] [Order article via Infotrieve]
  51. Agre, P., King, L. S., Yasui, M., Guggino, W. B., Ottersen, O. P., Fujiyoshi, Y., Engel, A., and Nielsen, S. (2002) J. Physiol. (Lond.) 542, 3–16[Abstract/Free Full Text]
  52. Verkman, A. S. (2005) J. Cell Sci. 118, 3225–3232[Abstract/Free Full Text]
  53. Nicchia, G. P., Frigeri, A., Nico, B., Ribatti, D., and Svelto, M. (2001) J. Histochem. Cytochem. 49, 1547–1556[Abstract/Free Full Text]
  54. Elkjaer, M., Vajda, Z., Nejsum, L. N., Kwon, T., Jensen, U. B., Amiry-Moghaddm, M., Frokiaer, J., and Nielsen, S. (2000) Biochem. Biophys. Res. Commun. 276, 1118–1128[CrossRef][Medline] [Order article via Infotrieve]
  55. Kishida, K., Shimomura, I., Kondo, H., Kuriyama, H., Makino, Y., Nishizawa, H., Maeda, N., Matsuda, M., Ouchi, N., Kihara, S., Kurachi, Y., Funahashi, T., and Matsuzawa, Y. (2001) J. Biol. Chem. 276, 36251–36260[Abstract/Free Full Text]
  56. Carbrey, J. M., Gorelick-Feldman, D. A., Kozono, D., Praetorius, J., Nielsen, S., and Agre, P. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 2945–2950[Abstract/Free Full Text]
  57. Matsuzaki, T., Suzuki, T., Koyama, H., Tanaka, S., and Takata, K. (1999) J. Histochem. Cytochem. 47, 1275–1286[Abstract/Free Full Text]
  58. Ma, T., Hara, M., Sougrat, R., Verbavatz, J.-M., and Verkman, A. S. (2002) J. Biol. Chem. 277, 17147–17153[Abstract/Free Full Text]
  59. Anthony, T. L., Brooks, H. L., Boassa, D., Leonov, S., Yanochko, G. M., Regan, J. W., and Yoo, A. J. (2000) Mol. Pharmacol. 57, 576–588[Abstract/Free Full Text]
  60. Engel, A., Fujiyoshi, Y., and Agre, P. (2000) EMBO J. 19, 800–806[CrossRef][Medline] [Order article via Infotrieve]
  61. Ohishi, Y., Oda, Y., Uchiumi, T., Kobayashi, H., Hirakawa, T., Miyamoto, S., Kinukawa, N., Nakano, H., Kuwano, M., and Tsuneyoshi, M. (2002) Clin. Cancer Res. 8, 3767–3775[Abstract/Free Full Text]
  62. Nakamura, T., Sakaeda, T., Ohmoto, N., Tamura, T., Aoyama, N., Shirakawa, T., Kamigaki, T., Nakamura, T., Kim, K. I., Kim, S. R., Kuroda, Y., Matsuo, M., Kasuga, M., and Okumura, K. (2002) Drug Metab. Dispos. 30, 4–6[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Toxicol SciHome page
D. S. Miller, J. R. Shaw, C. R. Stanton, R. Barnaby, K. H. Karlson, J. W. Hamilton, and B. A. Stanton
MRP2 and Acquired Tolerance to Inorganic Arsenic in the Kidney of Killifish (Fundulus heteroclitus)
Toxicol. Sci., May 1, 2007; 97(1): 103 - 110.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow An addition or correction has been published
Right arrow All Versions of this Article:
281/27/18401    most recent
M601266200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lee, T.-C.
Right arrow Articles by Huang, J.-d.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lee, T.-C.
Right arrow Articles by Huang, J.-d.
Social Bookmarking
Add to CiteULike   Add to Complore