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[Cancer Research 61, 7552-7555, October 15, 2001]
© 2001 American Association for Cancer Research

Experimental Therapeutics

Cross-Resistance to the Synthetic Retinoid CD437 in a Paclitaxel-resistant Human Ovarian Carcinoma Cell Line Is Independent of the Overexpression of Retinoic Acid Receptor-{gamma}1

Atul Kumar, Dianne R. Soprano and Hemant K. Parekh2

Departments of Pathology and Laboratory Medicine [H. K. P.] and Biochemistry [D. R. S.] and Fels Institute for Cancer Research & M. D./Ph. D. Program [A. K.], Temple University School of Medicine, Philadelphia, Pennsylvania 19140


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Treatment of ovarian carcinomas with the antimitotic antitumor drug paclitaxel is highly efficacious. However, development of drug resistance presents a major obstacle. The common cellular phenotypes associated with paclitaxel resistance are an increased expression of the drug transport protein P-glycoprotein (P-gp), an alteration in the levels of ß-tubulin isotypes, and/or changes in the drug binding affinity of the microtubules. We established two paclitaxel-resistant human ovarian carcinoma cell lines. The 2008/17/4 cells exhibited a "classic" multidrug-resistant phenotype (overexpression of P-gp associated with cross-resistance to natural product drugs), whereas the 2008/13/4 cells were an atypical multidrug-resistant subline (no overexpression of P-gp). In addition to being paclitaxel resistant (250-fold), the 2008/13/4 cells were also cross-resistant to etoposide (39-fold) and vincristine (460-fold). To identify the alterations in the gene expression profile associated with the development of atypical paclitaxel resistance, we used the Clontech Atlas Human Cancer cDNA Microarray (spotted with 588 genes). The expression of retinoic acid receptor (RAR)-{gamma} was significantly higher in the paclitaxel-resistant (2008/13/4 and 2008/17/4) cells than in the parental (2008) cells. Northern blotting analysis demonstrated that the expression of RAR-{gamma} was 7-fold higher in the 2008/13/4 and 2008/17/4 cells than in the 2008 cells, whereas the expression of RAR-{alpha} and RAR-ß was not observed in any cell line. Whereas the 2008, 2008/13/4, and 2008/17/4 cells were found to resist the antiproliferative effects of all-trans-retinoic acid, the paclitaxel-resistant cells were 6- to 7-fold cross-resistant to the antiproliferative effects of CD437 (a synthetic RAR-{gamma}-selective agonist; 6-[-(1-admantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid) compared with the sensitivity of the parental cells. To further understand the association of paclitaxel and CD437 resistance with the observed RAR-{gamma} overexpression, we transfected the 2008 cells with a full-length RAR-{gamma} cDNA construct. Two transfectants with increased expression of the RAR-{gamma} mRNA and protein were isolated and subjected to growth inhibition assays in the presence of various concentrations of paclitaxel, etoposide, vincristine, and CD437. The sensitivity of the 2008 transfected clones (displaying increased expression of RAR-{gamma}) to the cytotoxic effects of paclitaxel, etoposide, vincristine, and CD437 was similar to that observed in the parental 2008 cells. These results suggest that the overexpression of RAR-{gamma} (observed in the 2008/13/4 and 2008/17/4 cells) by itself is not capable of inducing paclitaxel and CD437 resistance (or resistance to etoposide and vincristine).


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Paclitaxel, an antimitotic anticancer drug, has a unique mechanism of action: it binds to ß-tubulin and induces the formation of highly stable microtubules that resist depolymerization (1 , 2) . This stabilization of microtubules causes cell cycle arrest and eventually causes cell death (1) . Paclitaxel is highly efficacious in the treatment of a variety of solid tumors as well as hematological malignancies (3) . However, development of tumor cell resistance to the cytotoxic effects of paclitaxel limits its therapeutic efficacy. Furthermore, paclitaxel resistance is often associated with development of cross-resistance to multiple structurally and functionally dissimilar drugs, which is likely attributable to the overexpression of P-gp,3 a transmembrane protein involved in transport that reduces the intracellular concentration of the drugs (4 , 5) . In contrast, P-gp-negative paclitaxel-resistant cells display an alteration in the expression or drug binding affinity of the tubulins and are cross-resistant to drugs targeted against the microtubules but not to other DNA-interactive anticancer drugs (4 , 5) .

We isolated two paclitaxel-resistant human ovarian carcinoma cell lines, one of which (2008/17/4) exhibits a "classical" multidrug-resistant phenotype associated with increased expression of P-gp and cross-resistance to etoposide, vincristine, and Adriamycin. In contrast, the 2008/13/4 cells are cross-resistant to etoposide and vincristine (but not to Adriamycin and cisplatin; Ref. 6 ). Furthermore, the drug resistance phenotype of these cells (2008/13/4) is not associated with the overexpression of P-gp or changes in the tubulin isotype or drug binding affinity of the microtubules (6) . To identify the genetic changes leading to this atypical resistance phenotype, we analyzed the expression pattern of 588 genes using a cDNA microarray.

RAR-{gamma} was found to be overexpressed in the paclitaxel-resistant cells. Furthermore, the 2008/13/4 and 2008/17/4 cells exhibited a 6- to 7-fold cross resistance to the antiproliferative effects of CD437 (6-[-(1-admantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid), a RAR-{gamma}-selective synthetic retinoid. Constitutive overexpression of RAR-{gamma} in the parental (2008) human ovarian carcinoma cells did not lead to concomitant induction of paclitaxel or CD437 resistance. These results suggest that the CD437 cross-resistance in the paclitaxel-resistant human ovarian carcinoma cells is not associated with the observed overexpression of RAR-{gamma}.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Materials.
Paclitaxel was obtained from the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute (NIH, Bethesda, MD). Paclitaxel was dissolved in DMSO at a final concentration of 20 mM, and the concentration of the solvent never exceeded 0.1% in the experimental protocol. ATRA was kindly supplied by Hoffmann-La Roche (Nutley, NJ) and prepared as a 10-3 M stock solution in ethanol. CD437 (a RAR-{gamma}-selective agonist; Ref. 7 ) was kindly supplied by Galderma and prepared as a 10-3 M stock solution in DMSO. All procedures using retinoids were carried out under subdued light. Full-length mouse RAR-{gamma} was obtained as a generous gift from Prof. Pierre Chambon (Institut de Genetique et de Biologie Moleculaire et Cellulaire, Strasburg, France). RAR-{gamma} mouse monoclonal antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). MTT was purchased from Sigma Chemical Co. (St. Louis, MO). [{alpha}-32P]dCTP (3000 Ci/mmol) was purchased from DuPont-New England Nuclear Research Products (Boston, MA). The multiprime DNA labeling system was obtained from Amersham Corp. (Arlington Heights, IL).

Cell Culture Conditions.
The parental (2008) and paclitaxel-resistant (2008/13/4 and 2008/17/4) human ovarian carcinoma cells were maintained as described previously (6) . The 2008/13/4 cells and 2008/17/4 cells were obtained after exposure of the parental 2008 cells to stepwise increasing concentrations of paclitaxel. Details of the procedure used to establish the paclitaxel-resistant cells have been described previously (6) .

Analysis of Differential Gene Expression Using cDNA Arrays.
Atlas Human Cancer cDNA Expression Array (catalog number 7740-1; Clontech, Palo Alto, CA) was used to analyze the differential gene expression in the parental (2008) and paclitaxel-resistant (2008/13/4) cells. Isolation of total RNA, cDNA synthesis, labeling of cDNA, and hybridization of cDNA probes to the Atlas Array filters were performed according to the manufacturer’s protocol (Clontech). After hybridization and washing, the array filters were scanned using a Fuji phosphorimager and quantitated using MacBAS (Fuji) software.

Northern Blotting.
The presence of the differentially expressed cDNA observed in the cDNA expression arrays was verified by Northern blotting using a full-length RAR-{gamma} cDNA as a probe that was radiolabeled using the random priming labeling method. Northern blotting was performed as described previously (6) .

Growth Inhibition Studies.
The sensitivity of parental 2008 and paclitaxel-resistant 2008/13/4 and 2008/17/4 cells to various anticancer drugs and retinoids was assessed using the MTT method as described previously (6) . The IC50 for each cell line was calculated by linear regression analysis. Each of the cell lines was exposed to varying concentrations of drugs in triplicate, and each experiment was repeated at least three times.

Transfection of the Full-length RAR-{gamma} cDNA in 2008 Cells.
Subconfluent cells (2 x 105 cells/60-mm dish) were transfected with pOPRSV vector (Stratagene, La Jolla, CA) alone or the pOPRSV vector containing the full-length RAR-{gamma} insert (2 µg/ml) using the LipofectAMINE reagent (Life Technologies, Inc., Gaithersburg, MD). The construction of the vectors has been reported previously (8) . Cells were propagated in medium containing Geneticin (700 µg/ml; G418 sulfate) for 3 weeks. Individual G418-resistant colonies were picked, propagated, and screened for the constitutive expression of RAR-{gamma} by Northern blotting and Western blotting.

Western Blotting.
Nuclear proteins from the parental (2008), paclitaxel-resistant (2008/13/4), empty vector-transfected (2008/mock), and RAR-{gamma}-transfected (2008/RAR2 and 2008/RAR3) cells were extracted as described previously (9) . Briefly, cells were plated at a density of 5 x 106 cells/dish and allowed to grow overnight in growth media. To isolate the nuclear proteins, the cells were pelleted, washed three times with chilled PBS, pelleted, and resuspended in ice-cold buffer (Buffer A) containing 10 mM HEPES (pH 7.8), 10 mM KCl, 2 mM MgCl2, 1 mM DTT, 0.1 mM EDTA, and 1x protease inhibitor mixture. After centrifugation at 2,500 rpm for 10 min, the cell pellet was resuspended in Buffer A containing 1% NP40 and mixed vigorously for 15 s. The homogenate was then centrifuged at 14,000 rpm for 30 s to separate the nuclei (pellet fraction) from the cytosol (supernatant). The nuclear pellet was washed once with Buffer A and then resuspended in ice-cold buffer containing 50 mM HEPES (pH 7.8), 50 mM KCl, 300 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 1x protease inhibitor mixture, and 10% glycerol and mixed for 20 min on an orbital shaker. The lysate was then centrifuged at 14,000 rpm for 5 min, the supernatant containing the nuclear proteins was collected, and aliquots were frozen immediately in dry ice and stored at -80°C until use.

Nuclear proteins (20 µg) were fractionated by SDS-PAGE and transferred to polyvinylidene difluoride membrane using a semidry blotter. After 1 h of incubation in blocking buffer containing TBS-T and 5% nonfat dry milk (w/v), the membrane was incubated overnight at 4°C with RAR-{gamma}-specific mouse monoclonal antibody (1 µg/10 ml TBS-T), washed three times for 5 min with TBS-T, incubated for 1 h at room temperature with secondary antibody coupled to horseradish peroxidase (diluted 1:5000) in TBS-T, and washed three times for 5 min in TBS-T. Specific binding of the primary antibody was visualized using the enhanced chemiluminescence detection kit (Pierce, Rockford, IL).

Statistical Analysis.
The linear regression analysis was performed using the SigmaStat Statistical Analysis System, Version 1.01.


   RESULTS AND DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
We established two paclitaxel-resistant cell lines designated 2008/13/4 and 2008/17/4 from a human ovarian carcinoma cell line (2008; Ref. 6 ). The 2008/17/4 cells were >1500-fold resistant to paclitaxel and display cross-resistance to several natural product drugs (6) . The multidrug-resistant nature of these cells has been associated with an increased expression of the P-gp (a drug efflux pump) leading to a significant decrease in the intracellular accumulation of paclitaxel (6) . In contrast, the 2008/13/4 cells were 252-fold paclitaxel resistant and expressed no P-gp (as assessed by Western blotting) or very low levels of P-gp (as assessed by reverse transcription-PCR) and had no obvious alterations in the expressions of various tubulin isotypes or defects in the ability of the microtubules to bind paclitaxel (6) . Furthermore, the intracellular levels of paclitaxel in the 2008/13/4 cells (after a 4-h exposure to 5.16 nM paclitaxel) were similar to those observed in the parental 2008 cells, thus confirming that P-gp is not involved in the development of paclitaxel resistance in the 2008/13/4 cells (6) . These observations suggest that the differential expression of hitherto unknown effectors was responsible for the development of resistance in the 2008/13/4 cells. To catalogue these changes at the molecular level, it was necessary to use a high throughput assay that would enable us to globally compare the pattern of cellular gene expression in the parental and paclitaxel-resistant cells.

The recently introduced cDNA expression microarray technique is one such method that would allow measurement of temporal changes in the gene expression profiles during the development of paclitaxel resistance. Indeed, this methodology has been used successfully (10) to analyze alterations in the gene expressions in doxorubicin-sensitive and -resistant tumor cells. In this study, we used the human cancer cDNA microarray spotted with 588 genes (Clontech). Several genes were identified that were differentially expressed in the 2008/13/4 cells as compared with their expression in the 2008 cells (Fig. 1Citation , arrows; Table 1Citation ).



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  		Fig. 1. Gene expression profile of parental (2008) and paclitaxel-resistant (2008/13/4) cells. Atlas Human cDNA Expression Arrays were used to analyze the differential gene expression of 2008 and 2008/13/4 cells. The arrays include 588 human cDNAs, 9 housekeeping control cDNAs, and negative controls in duplicate immobilized on a nylon membrane. Increased expression of genes in either the 2008 or 2008/13/4 cells is indicated by arrows. Boxes highlight the position and expression of RAR-{gamma}. These boxes are further magnified and displayed as insets below each array.

 

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  		Table 1 Identity and GenBank accession of the differentially expressed genes (see Fig. 1Citation ) from the parental (2008) and Taxol-resistant (2008/13/4) cells

 
Expression of two negative regulators of metastasis (NM23-H2 and NM23-H4; Refs. 11 and 12 ) was decreased in the paclitaxel-resistant 2008/13/4 cells. In addition, expression of EGR1, an early growth response gene known to modulate the expression of P-gp (13) , was lower in the paclitaxel-resistant cells (Table 1)Citation . It can be speculated that the decreased expression of P-gp observed in the 2008/13/4 cells could be associated with the down-regulation of EGR1 expression. In the 2008/13/4 cells, expression of cyclin-dependent kinase 10, p16 (an inhibitor of cyclin-dependent kinase 4), integrins {alpha}7 and ß4, low density lipoprotein-related protein 1, a small GTP-binding protein homologous to Rac2, and RAR-{gamma} was higher than that in the 2008 cells (Table 1)Citation . At present, it is not known whether the differential expression of each of these genes is directly associated with the development of paclitaxel resistance; investigation in this matter is under way.

Germane to this study was the observed overexpression of the RAR-{gamma} mRNA (Fig. 1Citation , inset) in the 2008/13/4 cells as compared with that observed in the 2008 cells.

To confirm the differential expression observed in the cDNA microarray analysis, Northern blotting analysis was performed using a full-length RAR-{gamma} cDNA. As shown in Fig. 2Citation , the expression of RAR-{gamma} was 7-fold higher in the 2008/13/4 and 2008/17/4 cells than in the 2008 cells. In contrast, expression of RAR-{alpha} and RAR-ß was not observed by Northern blotting analysis in either cell line (data not shown).



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  		Fig. 2. Expression of RAR-{gamma} mRNA in the 2008 (Lane 1), 2008/13/4 (Lane 2), and 2008/17/4 (Lane 3) cells. A, Northern blot analysis of total RNA extracted from the parental and paclitaxel-resistant cells using a full-length mouse RAR-{gamma} cDNA probe. The arrow indicates the position of the RAR-{gamma} message in each cell line. B, as a control for RNA loading, RNA gels were stained with ethidium bromide to compare the levels of 18S and 28S rRNA.

 
The RARs are a family of nuclear receptors that upon activation (through binding of retinoic acid) dimerize with one of the RXR subfamily of receptors and then bind to a regulatory element, retinoic acid response element, on the target genes and modulate their expression (14) . Three distinct subtypes of the RARs and RXRs have been identified, i.e., RAR-/RXR-{alpha}, -ß, and -{gamma} (14) . Expression of specific RAR or RXR subtypes has been demonstrated to affect the antiproliferative effects of natural as well as synthetic retinoids (14) . Inducible overexpression of RAR-{gamma} and constitutive expression of RXR-{alpha} in a human ovarian carcinoma cell line resistant to ATRA led to increased sensitivity to the apoptotic effects of CD437 (8) . Furthermore, CD437 has been demonstrated to induce apoptosis and cell death in a wide variety of tumor cell types, and these effects have been shown to be dependent as well as independent of the expression of RAR-{gamma} (14) .

Thus, we wished to analyze whether the up-regulation of RAR-{gamma} would affect the sensitivity of the 2008/13/4 cells to retinoids. The 2008, 2008/13/4, and 2008/17/4 cells were resistant to the growth-inhibitory effects of ATRA across a wide range of concentrations studied (10-10 to 10-6 M; data not shown). In contrast, the 2008/13/4 cells were found to be 6-fold resistant (IC50 = 4.5 µM) to the antiproliferative effects of CD437, and the 2008/17/4 cells were 7-fold resistant (IC50 = 5.2 µM) to the antiproliferative effects of CD437 compared with the 2008 cells (IC50 = 0.74 µM; Fig. 3BCitation ). The degree of CD437 cross-resistance (6- to 7-fold) observed in the 2008/13/4 and 2008/17/4 cells indicates that this phenotype is not dependent on the level of P-gp expression.



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  		Fig. 3. Effect of paclitaxel (A) and CD437 (B) on the growth of 2008, 2008/13/4, and 2008/17/4 cells. The 2008 (•), 2008/13/4 ({circ}), and 200817/4 cells ({blacktriangleup}) were treated with various concentrations of Taxol (A) and CD437 (B), and after 72 h, growth was assessed by MTT assay (6) . Values, means ± SD of triplicate samples from three individual experiments.

 
To determine whether the resistance of the 2008/13/4 and 2008/17/4 cells to CD437 was directly associated with the overexpression of RAR-{gamma} observed in these cells, we analyzed the effects of CD437 against 2008 cells transfected with a full-length RAR-{gamma} cDNA. The total RNA from the transfectants (as assessed by their ability to grow in the presence of 700 µg/ml G418) was initially subjected to Northern and Western blotting to identify clones that constitutively overexpressed RAR-{gamma} (Fig. 4, A and B)Citation . Two clones were identified and subjected to growth inhibition assay in the presence of different concentrations of paclitaxel, etoposide, vincristine, ATRA, and CD437 (Table 2)Citation . The sensitivity of RAR-{gamma}-overexpressing clones to each of these drugs was found to be similar to the sensitivity of the parental 2008 cells and mock-transfected (empty vector-transfected) 2008 cells (Table 2)Citation . Inducible overexpression of RAR-{gamma} has been reported to increase the apoptotic cell death in response to CD437 in ATRA-resistant human ovarian carcinoma cells (SK-OV3 cells; Ref. 8 ), and this observation is in contrast to our results, in which overexpression of RAR-{gamma} was unable to modulate the CD437 effects against the 2008 human ovarian carcinoma cells. An explanation for this differential effect of RAR-{gamma} overexpression on CD437 effects is not available but is most likely attributable to the different tumor cell lines studied. Moreover, although CD437 is known to be a RAR-{gamma}-selective retinoid that specifically binds to and transactivates RAR-{gamma} (7) , the suppression of tumor cell growth and induction of apoptosis by CD437 seem to be unrelated to the expression of RAR-{gamma} (14) .



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  		Fig. 4. Constitutive expression of RAR-{gamma} in 2008 cells transfected with a full-length mouse RAR-{gamma} cDNA. A, total RNA was isolated from the RAR-{gamma}-transfected single-cell clones (2008/RAR2 and 2008/RAR3), empty vector-transfected 2008 cells (2008/mock), and parental 2008 cells and subjected to Northern blotting. The position of the endogenous and recombinant RAR-{gamma} transcript is indicated. B, Western blot analysis of the RAR-{gamma} protein expression in 2008/mock cells (Lane 1), 2008/RAR2 RAR-{gamma}-transfected single-cell clones (Lane 2), 2008/RAR3 cells (Lane 3), parental 2008 cells (Lane 4), and paclitaxel-resistant 2008/13/4 cells (Lane 5). Nuclear proteins were extracted as described in "Materials and Methods," and 20 µg from each cell line were subjected to Western blotting analysis using a mouse monoclonal antibody against RAR-{gamma}. Specific bands were visualized by enhanced chemiluminescence as described in "Materials and Methods."

 

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  		Table 2 The effect of constitutive overexpression of RAR-{gamma} in the 2008 cells on the cytotoxicity of paclitaxel, etoposide, vincristine and CD437

 
Interestingly, the reported biochemical and cellular alterations in response to CD437 exposure are very similar to those observed upon treatment of tumor cells with paclitaxel (15, 16, 17, 18, 19, 20, 21, 22, 23) . Thus, paclitaxel has also been shown to be effective against cisplatin-refractory ovarian carcinomas (15) . Similarly, cisplatin-resistant ovarian tumors have been shown, both in vitro and in vivo, to be responsive to CD437 (16) . Treatment with CD437 and paclitaxel has been shown to up-regulate the expression of the cyclin-dependent kinase inhibitor p21waf1/cip1 (17, 18, 19, 20) . Furthermore, both paclitaxel and CD437 induce p53-dependent and -independent apoptosis in human cancer cells (17, 18, 19, 20) . Overexpression of the antiapoptotic bcl-2 inhibits both CD437- and paclitaxel-induced apoptosis (17 , 21) . In tumor cells undergoing apoptosis attributable to treatment with CD437 and paclitaxel, increased expression of the DNA damage-inducible protein GADD45 has been demonstrated (17 , 22) . Furthermore, activation of the p38 MAPK and the c-Jun NH2-terminal kinase/stress-activated protein kinase pathway during apoptosis in response to paclitaxel and CD437 treatment has been observed (17 , 23) . In this regard, we have recently demonstrated that paclitaxel is unable to activate the p38 MAPK in 2008/13/4 cells.4 It would be interesting to ascertain whether CD437 also lacks the ability to modulate the activity of p38 MAPK in 2008/13/4 cells. Lack of p38 MAPK activation (a phenomenon that induces apoptosis) by either paclitaxel or CD437 would elucidate a common cellular pathway that could account for the cross-resistance pattern of the paclitaxel-resistant cells.

Our results thus far indicate that development of resistance to paclitaxel renders a human ovarian carcinoma cell cross-resistant to the cytotoxic effects of CD437. Whereas the cross-resistance to CD437 is not associated with the level of RAR-{gamma} expression in the paclitaxel-resistant cells, the similar profile of biochemical alterations induced by paclitaxel and CD437, albeit in different systems, lends credence to our notion that a common pathway responsible for the antiproliferative effects of both CD437 and paclitaxel is disrupted in the paclitaxel-resistant human ovarian carcinoma cells. Thus, cross-resistance could potentially be associated with alterations in any of the aforementioned cellular effectors of paclitaxel and/or CD437 toxicity, or it could be related to some other novel pathway. Considering the potential utility of this new and unique class of synthetic retinoids in the chemotherapeutic treatment of human cancer, the paclitaxel-resistant human ovarian carcinoma cells would be a useful tool in the elucidation of the molecular mechanism underlying the development of resistance to CD437 as well as to paclitaxel.


   FOOTNOTES
 
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.

1 Supported by NIH Grant RO1-CA76400 (to H. K. P.). Back

2 To whom requests for reprints should be addressed, at Department of Pathology and Laboratory Medicine, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140. Phone: (215) 707-8024; Fax: (215) 707-2781; E-mail: hparek00{at}thunder.temple.edu Back

3 The abbreviations used are: P-gp, P-glycoprotein; RAR, retinoic acid receptor; ATRA, all-trans-retinoic acid; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; RXR, retinoic acid X receptor; MAPK, mitogen-activated protein kinase; TBS-T, 25 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.05% Tween 20 (v/v). Back

4 H. Tatineni and H. K. Parekh, unpublished observation. Back

Received 5/ 9/01. Accepted 8/15/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 

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