ARTICLE |
Correspondence to: Jeanne-Marie Bréchot, Hôtel-Dieu, 1 Place du Parvis Notre-Dame, 75181 Paris Cedex 04, France.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The MRP (multidrug resistance-associated protein) transmembrane transporter, which actively transports a wide variety of lipophilic substrates out of cancer cells, has been suggested to play a major role in cell detoxification via efflux of glutathione conjugates. Because bronchial epithelial cells are constantly exposed to environmental pollutants, MRP might be a particularly important defense mechanism against xenobiotics. This study was therefore designed to investigate MRP localization by immunohistochemistry in bronchial epithelial cells collected by scraping from surgical specimens. In parallel, MRP mRNA was detected by reverse transcriptase chain reaction (rt-PCR) in bronchial cell lysates. However, the pattern of protein expression differed markedly according to cell type. In ciliated epithelial cells, immunostaining was restricted to the basolateral surface, without any labeling at the apical surface, which is at variance with the localization of CFTR and MDR1 proteins, other members of the same family of transporters. In basal cells, MRP was present over the entire circumference of the plasma membrane. Basal cells were identified by their morphology and specifically after incubation with an anticytokeratin 17 monoclonal antibody. In conclusion, the different patterns of localization suggest specific roles for MRP in basal and ciliated cells. (J Histochem Cytochem 46:513517, 1998)
Key Words: MRP, bronchial epithelium, basal cells, ciliated cells
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
TRANSMEMBRANE TRANSPORT, mediated by specific membrane-associated proteins, is responsible for a wide range of functions (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bronchial Epithelial Cells
Normal bronchial epithelial cells were collected by gentle scraping of the lumen of a normal main or lobar bronchus of a surgical specimen. These specimens were collected in 21 patients (15 men, six women) who underwent pulmonary resection for primary or secondary lung carcinoma. Thirteen were current smokers, five were ex-smokers who had quit smoking for more than 2 years before surgery, and three were non-smokers. The bronchial section was previously examined and considered to be histologically normal. The quality of the samples was controlled by phase-contrast microscopy showing more than 97% epithelial cells, predominantly basal cells and ciliated cells (viable as assessed by their ciliary movements). Cytocentrifuge preparations were made when the material was sufficient (n = 11) and were stored at -80C until immunocytochemistry (ICC).
Identification of basal cells was performed by ICC using a specific anti-human cytokeratin 17 MAb (Dako; Glostrup, Denmark) as previously reported (
T47D cells from a breast carcinoma cell line, purchased from the American Type Culture Collection (Rockville, MD), were used as positive controls for rt-PCR and ICC. We have previously checked, using rt-PCR and ICC, that T47D cells express MRP in the same way as MCF7 cells, another breast cancer cell line (
Evaluation of MRP mRNA Expression by rt-PCR
Preparation of cDNA.
Bronchial epithelial cells were pelleted by centrifugation. Total RNA was isolated by acid guanidine thiocyanatephenolchloroform as previously described (
PCR Amplification. For PCR, 5 µl of the cDNA mixture was amplified in a 25-µl reaction volume containing 2.5 mM MgCl2, 0.5 mM of each dNTP, 0.5 U Taq polymerase (ATGC Biotechnologie; Noisy-le-Grand, France), and 37.5 pmol of sense-strand and antisense-strand MRP primers. ß2-Microglobulin (ß2m) cDNA was co-amplified in the same reaction with 20 pmol of each ß2m primer to control the quality of the cDNA and for quantification. Thermal cycling was performed on a Kontron thermoblock (Biometra-Trio-Thermoblock; Tampa, FL). After an initial denaturation at 95C for 5 min, each sample underwent 30 cycles of denaturation at 95C for 1 min, annealing at 55C for 1 min, and extension at 72C for 1 min. Final extension at 72C lasted 7 min. In a preliminary experiment using a range from 10 to 40 cycles for amplification, we confirmed that 30 cycles was still in the exponential phase for MRP as well as for ß2m (results not shown). Negative control reactions were included by omitting RNA, reverse transcriptase, or cDNA template. PCR products were separated on a 2% agarose gel containing ethidium bromide (Bioprobes Systems; Montreuil sous Bois, France).
Primers.
MRP primers (Oligo Express; Paris, France) were designed according to previously published sequences (
Quantification of the PCR Products. Semiquantitative rt-PCR analysis was performed on photographs from ethidium bromide-stained agarose gels, scanned and analyzed with a computerized image analysis system (Biorad Imaging Densitometer GS670; Ivry-sur-Seine, France). For each of the samples, MRP mRNA content was expressed as the ratio of the densitometry value of MRP in relation to that of ß2m.
Statistical Analysis. The number of samples with a positive MRP mRNA detection was compared in the two groups of current smokers on the one hand (n = 13), and ex- and non-smokers on the other hand (n = 8) using the Fisher's exact test. The results of MRP mRNA semiquantification obtained in each group were expressed as means and standard errors. The MannWhitney U-test was used to compare the mean ratio in the two groups. Statistical significance was set at p<0.05.
Evaluation of MRP Protein Localization by Immunocytochemistry
Anti-MRP Antibodies.
Two anti-MRP MAbs were tested for immunodetection of MRP protein: MC-201 and MC-202 (SyrinxDiagnostika; Frankfurt, Germany). Both are reported to recognize internal epitopes and not to crossreact with the human MDR1 and MDR3 P-glycoproteins (
Immunocytochemical Procedure. Conditions for immunodetection of MRP were evaluated using the T47D human breast cancer cell line. The two MAbs (MC-201 and MC-202) were used at various dilutions. The best immunostaining was obtained using MC-201 at a 1:40 dilution. MAb MC-201 was therefore used for MRP immunolocalization in bronchial epithelial cells.
Centrifuge preparations of bronchial epithelial cells collected by scraping were air-dried at room temperature for 12 hr and were fixed in acetone for 10 min at 4C. The slides were incubated for 20 min with normal rabbit serum (1:70; Vector, Burlingame, CA), followed by MAb MC-201 (1:40) incubation for 1 hr. MAb binding was detected using biotinylated rabbit anti-rat IgG (1:200) and avidinbiotin complex (1:50; Vector). Bound peroxidase was developed with diaminobenzidine tetrahydrochloride (Vector) and H2O2. The slides were counterstained with hemalum and mounted for examination.
Various negative controls were performed, omitting either the primary or the secondary antibody or substitution of the primary antibody by an isotype-matched rat non-immune IgG. T47D cells were used as positive controls.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Detection of MRP mRNA by rt-PCR in Bronchial Epithelial Cells
MRP mRNA was detected in 18 (86%) of the 21 samples tested. In a series of nine cases, two independent PCRs were performed in two different batches and their results were completely concordant. As illustrated in Figure 1, after reverse transcription and amplification with MRP-specific primers, a 290-BP band was detected in 11 of the 13 samples from smokers, in four of the five samples from ex-smokers, and in the three samples from non-smokers. Therefore, MRP mRNA was detected in 11 samples from the current smokers group (n = 13) and seven samples from the ex- and non-smokers group (n = 8). The densitometric evaluation of MRP mRNA contents is shown in Figure 2. No statistically significant difference was observed between the two groups (p>0.9). Negative results were observed in three samples (from two smokers and one ex-smoker). However, ICC was available in two of these three samples and showed the presence of MRP protein.
|
|
Immunolocalization of MRP Protein in Bronchial Epithelial Cells
Similar results were obtained in all of the 11 samples analyzed (from nine smokers and two ex-smokers).
Basal cells were clearly identified on the basis of their morphology and their intense labeling when incubated with the anti-CK 17 MAb (Figure 3A). After incubation with the anti-MRP MC-201 MAb, strong labeling was observed on the entire circumference of the plasma membrane (Figure 3B). No intracytoplasmic staining was observed.
|
Ciliated epithelial cells were identified by phase-contrast microscopy and by the absence of staining when incubated with the anti-CK17 MAb. After incubation with the anti-MRP MC-201 MAb (Figure 3D), the labeling pattern was different from that observed in basal cells. No immunostaining was detected at the apical region. In contrast, dense, continuous immunostaining was observed along the basolateral membrane of the cells. No intracytoplasmic staining was observed. Similar labeling was observed at the basolateral periphery of mucous cells.
Negative results were obtained when non-immune rat IgG was used instead of specific anti-MRP MAb (Figure 3C).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Analysis of the distribution of ABC proteins in bronchial epithelial cells constantly exposed to environmental pollutants is particularly important to the understanding of the mechanisms of resistance to xenobiotics. Indeed, transporters such as MDR1 Pgp or MRP exert various roles, providing a major contribution to cell detoxification.
In the present study, MRP mRNA was detected by rt-PCR in the majority of samples, in smokers as well as in non- or ex-smokers. Similar findings were previously reported in non-neoplastic bronchial epithelium, either by in situ hybridization (
This different pattern of distribution appears to be correlated with the existence of cell polarity. In basal cells with no tight junctions limiting apical and basolateral membrane domains, MRP was distributed over the entire circumference of the plasma membrane. In contrast, in polarized ciliated human bronchial cells MRP was restricted to the basolateral membrane. This pattern of expression has been previously reported in the polarized pig kidney epithelial cell line LLC-PK1 (
The different distribution of MDR1 Pgp, MRP, and CFTR in bronchial epithelial cells suggests specific functions for each one. MRP has been characterized as an ATP-dependent pump for endogenous and exogenous glutathione, glucuronate and sulfate conjugates (
![]() |
Acknowledgments |
---|
Supported by a grant from the Association Française de Lutte contre la Mucoviscidose 1994, France.
We thank M. Antoine, R. Lacave, S. Ricci, J. Rochemaure, and T. Urban for their constant support, V. Gerber for editorial assistance, and F. Stephan for statistics.
Received for publication July 15, 1997; accepted October 29, 1997.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abbaszadegan MR, Fuscher BW, Klimecki WT, List A, Dalton WS (1994) Analysis of multidrug resistance-associated protein (MRP) messenger RNA in normal and malignant hematopoietic cells. Cancer Res 54:4676-4679[Abstract]
Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 92:156-159
Cole SPC, Bhardwaj G, Gerlach JH, Mackie JE, Grant CE, Almquist KC, Stewart AJ, Kurz EU, Duncan AMV, Deeley RG (1992) Overexpression of a transporter gene in a multidrug resistant human lung cancer cell line. Science 258:1650-1654[Medline]
Crawford I, Maloney PC, Zeitlin PL, Guggino WB, Hyde SC, Turley H, Gatter KC, Harris A, Higgins CF (1991) Immunocytochemical localization of the cystic fibrosis gene product CFTR. Proc Natl Acad Sci USA 88:9262-9266[Abstract]
Evers R, Zaman GJR, Van Deemter L, Jansen H, Calafatj Oomen LCJM, Oude Elferink PJ, Borst P, Schinkel AH (1996) Basolateral localization and export activity of the human multidrug resistance-associated protein in polarized pig kidney cells. J Clin Invest 97:1211-1218
Flens MJ, Izquierdo MA, Scheffer GL, Fritz JM, Meijer CJLM, Scheper RJ, Zaman GJR (1994) Immunochemical detection of the multidrug resistance-associated protein MRP in human multidrug-resistance tumor cells by monoclonal antibodies. Cancer Res 54:4557-4563[Abstract]
Higgins CF (1992) ABC transporters: from microorganisms to man. Annu Rev Biol 8:67-113
Holtzman MJ (1991) Arachidonic acid metabolism. Implications of biological chemistry for lung function and disease. Am Rev Respir Dis 143:188-203[Medline]
Jedlitschky G, Leier I, Buchholz V, Barnouin K, Kurz G, Keppler D (1996) Transport of glutathione, glucuronate, and sulfate conjugates by the MRP gene-encoded conjugate export pump. Cancer Res 56:988-994[Abstract]
Jedlitschky G, Leier I, Bucholz U, Center M, Keppler D (1994) ATP-dependent transport of glutathione-S-conjugates by the multidrug resistance-associated protein. Cancer Res 54:4833-4836[Abstract]
LechaptZalcman E, Hurbain I, Lacave R, Commo F, Urban T, Antoine M, Milleron B, Bernaudin JF (1997) MDR1-PgP 170 expression in human bronchus. Eur Respir J 10:1837-1843
Leier I, Jedlitschky G, Buchholz U, Cole SPC, Deeley RG, Keppler D (1994) The MRP gene encodes an ATP-dependent export carrier for leukotriene C4 and structurally related conjugates. J Biol Chem 269:27807-27810
Nooter K, Bosman FT, Burger H, Van Wingerden KE, Flens MJ, Scheper RJ, Oostrum RG, Boersma AWM, Van der Gaast A, Stoter G (1996) Expression of the multidrug resistance-associated protein (MRP) gene in primary non-small-cell lung cancer. Ann Oncol 7:75-81[Abstract]
Puchelle E, Jacquot J., Fuchey C, Burlet H, Klossek JM, Gilain L, Triglia JM, Thinnes FP, Hilschmann N (1993) Immunolocalization of porin and CFTR channels in human surface respiratory epithelium. Biol Chem 374:297-304
Thomas GA, Barrand MA, Stewart S, Rabbitt PH, William ED, Twentyman PR (1994) Expression of the multidrug resistance-associated protein gene in human lung tumors and normal tissue as determined by in situ hybridization. Eur J Cancer 30A:1705-1709
Troyanovsky SM, Guelstein VI, Tchipysheva TA, Krutovskikh VA, Bannikov GA (1989) Patterns of expression of keratin 17 in human epithelia: dependency on cell position. J Cell Sci 93:419-426[Abstract]
Zaman GJR, Lankelma J, Van Tellingen O, Beijnen J, Dekker H, Paulusma C, Oude Elferink PJ, Baas F, Borst P (1995) Role of glutathione in the export of compounds from cells by the multidrug resistance-associated protein. Proc Natl Acad Sci USA 92:7690-7694[Abstract]
Zaman GJR, Versantvort CHM, Surit JJM, Eijdems EWHM, de Haas M, Smith AJ, Broxterman HJ, Mulder NH, de Vries EGE, Baas F, Borst P (1993) Analysis of the expression of MRP, the gene of a new putative transmembrane drug transporter, in human multidrug resistant lung cancer cell line. Cancer Res 53:1747-1750[Abstract]