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Am J Physiol Cell Physiol 295: C394-C405, 2008. First published June 11, 2008; doi:10.1152/ajpcell.00423.2007
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RECEPTORS AND SIGNAL TRANSDUCTION

Erythropoietin enhancement of rat pancreatic tumor cell proliferation requires the activation of ERK and JNK signals

Chhanda Bose1,3 and Kodetthoor B. Udupa1,2,3

1Donald W. Reynolds Departments of Geriatrics, 2Department of Physiology and Biophysics, University of Arkansas for Medical Sciences; and 3Medical Research, Central Arkansas Veterans Healthcare System, Little Rock, Arkansas

Submitted 14 September 2007 ; accepted in final form 7 June 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Erythropoietin (EPO) regulates the proliferation and differentiation of erythroid cells by binding to its specific transmembrane receptor EPOR. Recent studies, however, have shown that the EPOR is additionally present in various cancer cells and EPO induces the proliferation of these cells, suggesting a different function for EPO other than erythropoiesis. Therefore, the purpose of the present study was to examine EPOR expression and the role of EPO in the proliferation and signaling cascades involved in this process, using the rat pancreatic tumor cell line AR42J. Our results showed that AR42J cells expressed EPOR, and EPO significantly enhanced their proliferation. Cell cycle analysis of EPO-treated cells indicated an increased percentage of cells in the S phase, whereas cell numbers in G0/G1 phase were significantly reduced. Phosphorylation of extracellular regulatory kinase 1/2 (ERK1/2) and c-Jun NH2 terminal kinase 1/2 (JNK1/2) was rapidly stimulated and sustained after EPO addition. Treatment of cells with mitogen-activated protein/ERK kinase (MEK) inhibitor PD98059 or JNK inhibitor SP600125 significantly inhibited EPO-enhanced proliferation and also increased the fraction of cells in G0/G1 phase. Furthermore, the inhibition of JNK using small interference RNA (siRNA) suppressed EPO-enhanced proliferation of AR42J cells. Taken together, our results indicate that AR42J cells express EPOR and that the activation of both ERK1/2 and JNK1/2 by EPO is essential in regulating proliferation and the cell cycle. Thus both appear to play a key role in EPO-enhanced proliferation and suggest that the presence of both is required for EPO-mediated proliferation of AR42J cells.

erythropoietin receptor; cell signaling; mitogen-activated protein kinase induction

ERYTHROPOIETIN (EPO) is a glycoprotein hormone that plays a crucial role in erythropoiesis (39, 65). Its discovery and preparation through recombinant technology have made it available as a therapeutic drug. Moreover, with pure EPO available, its effect on erythroid cells has been determined to occur via its specific cell surface receptor EPOR (13). Recently, however, it has been reported that expression of EPOR is not restricted to hematopoietic cells but in a variety of other nonhematopoietic cells, such as endothelial, renal, neuronal, and cardiac cells in vitro (6, 58). Various cell types and tissues of human and animals also express EPO and EPOR (35), as do several malignant (25, 64, 70) and pediatric tumors (4). Finally, EPOR expression appears to play a role in tumor progression as suggested by several earlier studies (2, 3, 71), and hence, EPO may have a role beyond hematopoiesis.

EPO modulates a host of cellular signal transduction pathways to perform multiple functions in erythroid cells, as well as in other cellular systems (19, 48, 57). EPOR and EPO signaling is known to activate several intracellular kinase pathways, such as janus kinase 2 (JAK2)/signal transducer and activator of transcription (STAT), phosphatidylinositol-3-kinase (12, 57, 60), mitogen-activated protein kinases (MAPK) pathways, which include extracellular regulatory kinase 1/2 (ERK1/2), c-Jun NH2 terminal kinase 1/2 (JNK1/2), and p38 MAPK (14, 17, 29, 30, 49–51, 57, 66). MAPK represent a family of serine-threonine kinases involved in a wide range of cellular responses. Depending on the cellular context, activation of MAPK has been correlated with proliferation, differentiation, cell survival, and apoptosis (24, 40). Moreover, ERK1/2 is known to be induced by variety of growth factors and hormones, whereas JNK1/2 and p38 MAP kinases are activated by cellular stress, protein synthesis inhibitors, osmotic, heat, and chemical shock and are also thought to be associated with apoptosis (36, 41, 42, 44, 55, 56). Although JNK1/2 and p38 kinases are often linked to induction of apoptosis and stress, the actual roles of JNK1/2 and p38 are more complicated. It is now clear that both have very diverse roles in the regulation of cell proliferation and survival in some cell types, including erythroid cells (16, 31, 32). In addition, JNK1/2 has been shown to promote proliferation in response to platelet-derived growth factor and during liver regeneration (34, 61). Therefore, it is apparent that JNK1/2 also plays a role in mitogenic and growth factor signaling as well. Since the discovery of EPOR expression in cancer cells, there have been reports linking EPO-EPOR signaling in vitro to activate cancer and tumor cell proliferation (1–4, 21, 68, 71). However, relatively little is known about EPOR-induced signaling in tumor cells.

The aim of the current study was to show the presence of EPORs on rat pancreatic tumor (AR42J) cells and to determine the role of EPO in proliferation, as well as the mechanism of signal transduction involved in this process. Since the physiological and functional aspect of AR42J cells are similar to freshly isolated pancreatic acinar cells (10, 15), which we used in our previous studies on MAPK signaling (5), we elected to use this particular pancreatic tumor cell line in our current study.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. The AR42J cell line, derived from a rat pancreatic tumor, was obtained from the American Type Culture Collection (Rockville, MD). Cells were grown in 75-cm2 flasks in 12 ml of medium consisting of Ham's F-12 nutrient medium with 2 mM L-glutamine (Invitrogen Life Technologies, Carlsbad, CA), 1% penicillin and streptomycin, 1.5% sodium bicarbonate, and 10% fetal bovine serum (FBS, Cambrex Bioscience, Walkersville, MD). Flasks were placed in an incubator maintained at 37°C with 5% CO2 in air.

EPO treatment and inhibitor studies. For experiments with MAPK, 60–70% confluent cells were trypsinized, and 1.6 x 106 cells plated in 100-mm culture dishes in F-12 nutrient medium containing 10% FBS and allowed to attach for 12 h. On day 1, the medium was changed to serum-free nutrient medium. On day 2, cells were treated with various concentrations of EPO for different lengths of time as shown in RESULTS. Specific mitogen-activated protein (MAP)/ERK kinase (MEK) inhibitor PD98059, which acts by inhibiting the activation of MAP kinase (subsequently inhibiting phospho-ERK activation), and SP600125, a potent, cell-permeable JNK inhibitor (both from Calbiochem Biosciences, La Jolla, CA), were used in MAPK inhibitor studies. Cells were pretreated with 50 µM of PD98059 or 15 µM of SP600125 for 1 h before the addition of EPO. Equivalent volumes of either medium alone or medium containing DMSO were added to control cultures. The maximum concentration of DMSO in any experiment was 0.1% (vol/vol), which did not affect the activity or activation of any protein kinase examined. Human recombinant erythropoietin used in these studies was a generous gift from Centocor (a subsidiary of Johnson & Johnson), Malvern, PA.

Reverse-transcription polymerase chain reaction for EPOR expression. Cells were plated as described in the previous section, and total RNA was extracted using the RNeasy kit according to manufacturer's instructions (Qiagen, Valencia, CA). One microgram of total RNA from each sample was treated with 1 unit of RNase-free deoxyribonuclease I (Amplification Grade, Invitrogen) according to the protocol provided by the manufacturer. First DNA strand synthesis was carried out using 1 µg DNase-treated RNA and SuperScript II reverse transcriptase (Invitrogen) following the protocol provided by the manufacturer, except that a poly-dT16 primer was used. Mock cDNA preparation lacking reverse transcriptase was done in parallel for control reaction (no reverse transcriptase control). Reaction products (20 µl) were brought up to 200 µl with water and were stored at –20°C. For PCR amplification, 2 µl of cDNA was used in a 50-µl reaction volume containing Platinum PCR Super Mix High Fidelity (Invitrogen), with 0.3 µM of oligonucleotide primer specific for rat EPOR (sense: 5'-CTC TTA CCA GCT CGA AGG TGA-3' and antisense: 5'-TCC AGG ACC TCC ACC CTT TGT-3', synthesized by Integrated DNA Technology, Coralville, IA). PCR was performed by using GeneAmp PCR System 9600 (Perkin Elmer) following an initial incubation at 94°C for 2 min, the denaturation step was performed at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 68°C for 30 s for total 35 cycles. A final extension was performed at 68°C for 10 min after the last cycle. A negative control reaction was performed with water and mock cDNA preparation (no reverse transcriptase reaction). For positive control, mouse liver RNA was used. PCR products were analyzed by electrophoresis on 1.5% agarose gel. Amplified EPOR was gel purified and confirmed with DNA-sequencing analysis by automated sequencing at 3100 Genetic Analyzer (Applied Biosystems, Foster city, CA).

Cell proliferation assays. Proliferation of AR42J cells with various doses of EPO (1–10 mU/ml for 24–96 h) was accomplished by determining the bromodeoxyuridine (BrdU) incorporation during the last 4 h of incubation in the presence of 10 µM BrdU and measuring colorimetrically using the BrdU ELISA kit, (Roche Diagnostic, Indianapolis, IN), as described earlier (10). The effect of EPO on the proliferation of AR42J cells was also assessed by a high-sensitivity, commercial colorimetric assay WST-8 cell-counting kit (Dojindo Molecular Technologies, Gaithersburg, MD) using [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium] salt, as reported earlier (5, 10). In anti-EPOR antibody inhibition studies, AR42J cells were plated and treated with 5 mU/ml EPO for 48 h as described previously. Cells were pretreated with 0.02 µg/ml anti-EPOR antibody (H-194, sodium azide-free antibody purchased from Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h before the addition of EPO, and the proliferation was measured with the WST-8 cell-counting kit as described earlier.

Immunofluorescence study. AR42J cells were treated with 5 mU/ml of EPO for 5 and 30 min. For immunostaining, anti-ERK1/2, anti-JNK1/2, antiphospho-ERK1/2, and antiphospho-JNK-1/2 antibodies (Cell Signaling Technologies, Danvers, MA) and for EPOR anti-EPOR antibody (H-194, Santa Cruz Biotechnology, Santa Cruz, CA) were used with minor modification. AR42J cells grow in clumps and hence after being washed with cold PBS, cells were trypsinized and cytospun on to glass slides at 2,000 rpm for 2 min. The slides were fixed with 2% paraformaldehyde for 20 min at room temperature and then permealized with 1% Triton X-100 in PBS for 5 min. The remainder of the procedure was similar to that described earlier (5). For negative control, antigen blocking approach was used. For this experiment, 2 µg of rabbit polyclonal anti-EPOR (H-194) antibody was incubated with 18 µg of human recombinant EPOR protein (GenWay Biotech, San Diego, CA) in 0.2 ml volume of 0.1% human serum albumin in PBS overnight at 4°C. For immunofluorescence and Western blot analysis studies, the same concentration of antibody was used. In addition, for both, only secondary antibody-treated samples were used as additional negative controls.

Transfection of EPOR siRNA. For EPOR inhibition, a pool of three target-specific 20–25 nucleotide small interfering RNAs (siRNAs) for EPOR were commercially obtained from Santa Cruz Biotechnology (catalogue no. sc-77364). It had the following sequence: sense strand A, mRNA loc. 325-GCUCGAAGGUGAAUCAAGA; sense strand B, mRNA loc. 1179-CCUACCUGGUAUUGGAUGA; sense strand C, mRNA loc. 1270-GGAUGAAGGUUCAGAAACA, designed to knock down EPOR gene expression. Transfection protocol followed was as described by manufacturer. In brief, AR42J cells (2 x 106 cells/well) were seeded in 6-well tissue culture plate in 2 ml antibiotic free F-12 nutrient medium containing 10% FBS. Cells were incubated at 37°C overnight in an incubator with 5% CO2 in air. After 24 h, cells were washed with 2 ml transfection medium (sc-36868, Santa Cruz Biotechnology), and a mixture of 20–50 pmol of siRNA and 6 µl of transfection reagent (sc-29528, Santa Cruz Biotechnology) diluted in siRNA transfection medium was added to the washed cells. Cells were incubated for 7 h at 37°C in the incubator. One milliliter growth medium containing 2x serum and antibiotic was added to each well without removing the transfection mixture. After 12 h of incubation, the medium was replaced with 1x normal growth medium. Transfection efficiency was checked by real-time PCR, Western blot analysis, and immunoflourescence studies 24–48 h after the addition of fresh medium. For negative control siRNA-A (sc-37007) a nontargeted 20–25 nt siRNA was used. AR42J cells showed maximum knock down of the EPOR gene at 24 h with 25 pmol of siRNA (as examined by real-time PCR), and this condition and concentration were used for final experiments.

Flow cytometry analysis. After treatment of AR42J cells with 5 mU/ml EPO alone, MAPK inhibitors (50 µM of PD98059 or 15 µM of SP600125) alone, MAPK inhibitors and then EPO, or vehicle (0.1% DMSO) for 24 h to 96 h, the percentage of cells in the G0/G1, S, and G2/M phase of the cell cycle were determined by utilizing the Guava Cell Cycle reagent kit and performing flow cytometry using the Guava EasyCyte mini system. A total of 7,000 ungated events was acquired for each sample. Number of cells, shown under each histogram, represented at least 75% of all the acquired events. The data were further analyzed using CytoSoft software (Guava Technologies, Burlingame, CA), as described earlier (10).

Western blot analysis. After the treatment with EPO, samples were prepared and Western blot analysis was performed with antitotal and antiphospho antibodies of ERK-1/2, JNK-1/2, and p38 as described earlier (5, 10). The extent of phosphorylation was quantitated using a Strom 860 PhophoImager. For Western blot analysis studies of EPOR, 25 µg total proteins from each sample (AR42J cells, K562 cells) were electroblotted, and the membrane was blocked with 5% milk in TBS for 1 h at room temperature. For EPOR detection rabbit polyclonal anti-EPOR antibody (1:1,000, H-194, Santa Cruz, CA) was used in 0.5% milk in 1x TBST (TBS containing 0.1% Tween-20) for 2 h at room temperature followed by washing and incubation with horseradish peroxidase-conjugated secondary antibody, 1:2,000 dilution in 0.5% milk in TBST for 1 h at room temperature. Visualization of the bands was performed by utilizing SuperSignal West Pico Chemiluminescent substrate (Pierce, Rockford, IL), followed by radiography. Blots were stripped and reprobed with goat polyclonal anti-Actin (C-11). For negative control, antibody, H-194, incubated with excess antigen was used, as described in Immunofluorescence.

Kinase assays. Whole cell extracts from stimulated and nonstimulated cells were prepared by suspending cells in 500 µl of lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerol phosphate, 1 mM Na3VO4, and 1 µg/ml leupeptin) and sonicated on ice. After centrifugation at 14,000 g for 10 min at 4°C, supernatant was removed and used for kinase assays. ERK and JNK kinase assays were performed utilizing nonradioactive kinase assay kits (p44/42 MAP kinase assay kit for ERK1/2 activity and SAPK/JNK assay kit for JNK1/2 activity, both from Cell signaling Technologies, Danvers, MA) according to the manufacturer's instructions. ERK activity was measured by immune complex assay with GST-Elk-1 fusion protein (307–428) as substrate. Briefly, cell lysate (0.2 mg protein) was incubated overnight at 4°C with immobilized phospho-p44/42 antibody, centrifuged, and washed two times with ice-cold lysis buffer and two times with kinase buffer (in mM: 25 Tris, pH 7.5, 5 β-glycerol phosphate, 2 dithiothreitol, 0.1 Na3VO4, and 10 MgCl2). Pellet was resuspended with 50 µl of 1x kinase buffer supplemented with 200 µM of ATP and 2 µg of Elk-1 fusion protein. Reaction mixture was incubated for 30 min at 30°C. JNK1/2 activity assay was also performed in a similar manner except that a NH2-terminal c-Jun (1–89) fusion protein, bound to glutathione sepharose beads, was used to pull down JNK1/2 from the cell lysates. After the washings, pellets were resuspended in 50 µl of 1x kinase buffer supplemented with 200 µM of ATP and incubated for 30 min at 30°C. ERK1/2 and JNK1/2 activities were detected by SDS-PAGE and immunoblotting, using phospho-specific Elk-1 and c-Jun antibodies, respectively. Visualization and quantitation of phosphorylated proteins were performed by densitometry (Alpha Innotech, San Leandro, CA) or by scanning with Strom 860 Imager (Molecular Dynamics, Sunnyvale, CA).

Transfection of JNK siRNA. For JNK inhibition, chemically synthesized 25-nucleotide duplexes of Stealth siRNAs for JNK were commercially obtained from Invitrogen Life Technologies (Carlsbad, CA). We tried three different Stealth siRNA oligo duplexes targeting JNK. However, only one siRNA oligo duplex worked best, and the sequence was 5'-AUUACUAGGCUUUAAGUCCCGAUGA-3' and 5'UCAUCGGGACUUAAAGCCUAGUAAU-3'. AR42J cells were transfected with various concentrations of duplex Stealth siRNA (10, 25, and 50 nM), using 2 µg/ml of lipid-based transfection reagent (lipofectamine 2000, Invitrogen) for 72 h, according to the manufacturer's instructions. After 72 h of transfection, the cells were washed and treated with 5 mU/ml of EPO for a further 48 h. Proliferation was measured by WST-8 cell counting kit and the BrdU method as described in a previous section. For negative control, nonspecific siRNA with low GC content (Invitrogen) and lipofectamine 2000 were used. Inhibition of JNK1/2 expression was determined by immunoblotting samples of transfected cells in triplicate, as described earlier. To determine the efficiency of the oligonucleotide uptake, a fluorescein isothiocyanate-labeled antisense oligomer (BLOCK-iT, Invitrogen, at the concentration of 10, 20, and 50 nM) and lipofectamine 2000 (1 and 2 µg/ml) were used according to the manufacturer's instructions. Cells were examined under the microscope for fluorescence from 6 to 96 h after transfection. At 72 h, more than 60% of the cells displayed a relatively high fluorescence in cells transfected with the BLOCK-iT fluorescent oligomer at a concentration of 25 nM, and hence we used this concentration and time period for the transfection of AR42J cells with the duplex Stealth siRNA.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
EPOR expression in AR42J cells. We utilized four approaches to investigate whether AR42J cells exhibited EPOR on their surfaces. First, we performed immunostaining of AR42J cells grown exponentially in F-12 nutrient medium containing 10% FBS. As shown in Fig. 1A, AR42J cells showed a strong immunoreactivity toward EPOR when EPOR antibody was used. In the second approach, the transfection of siRNA for EPOR was done and it reduced this signal considerably indicating reduction of EPOR by this procedure (Fig. 1B). In the third approach, we performed RT-PCR using RNA from AR42J cells for EPOR. Thirty five cycles of RT-PCR were performed to visualize the EPOR PCR product at the expected size of 363 bp, the results of which are illustrated in Fig. 1E. The final product was also confirmed by direct sequencing for EPOR. In the forth approach, Western blot analysis was performed, which showed a ~66-kDa protein corresponding to EPOR when the total cell lysate of AR42J cells was used for the blot (Fig. 1F, lane 2). Transfection of siRNA for EPOR gene showed ~67% inhibition as examined by real-time quantitative PCR. Our Western blot analysis of control and transfected samples showed a ~41.4% suppression of EPOR on the protein level (Fig. 1F, between lanes 2 and 4). AR42J cells transfected with negative control has no effect on EPOR signals, in both immunofluorescence and Western blot analysis. EPOR band was also reduced when antigen-blocked antibody was used for Western blot analysis (Fig. 1F, lane 6). Hence, the detection of EPOR in RT-PCR studies, the reduction of staining in immunoflurescence, and reduction in band intensities on Western blots by siRNA transfection and immunoblot studies conform the presence of EPOR on AR42J cells.


Figure 1
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  		Fig. 1. Expression of erythropoietn (EPO) receptors (EPOR) on AR42J cells. A: immunofluorescence analysis of EPOR. AR42J cells were grown normally and trypsinized, cytospun as stated in MATERIALS AND METHODS and stained with anti-EPOR antibody (H-194), 1:100 dilution for 2 h followed by FITC-conjugated secondary antibody (1:100) for 1 h. They were counterstained with 4',6-diamino-2-phenylindole for nuclear staining. B: AR42J cells after transfection with small interfering RNAs (siRNA) for EPOR as described MATERIALS AND METHODS are shown. C: AR42J cells stained with secondary antibody alone (1:100) are shown. D: immunofluorescence study with AR42J cells treated with preincubated EPOR antibody with human recombinant EPOR protein are shown as described in MATERIALS AND METHODS. Fluorescence intensity was reduced very significantly in the cells treated with preincubated EPOR antibody. E: RT-PCR of RNA samples from AR42J cells were analyzed for EPOR expression. The positions of the 100-bp DNA ladder are shown in the lane marked M. As described in MATERIALS AND METHODS, EPOR-specific oligonucleotide primers were used to amplify 363 bp of EPOR cDNA from AR42J cells and depicted under the lane marked AR42J. As a positive control of the method, RT-PCR was performed using mouse liver RNA and depicted in the lane marked as ML. Lane 4, no RT-negative control; lane 5, water-negative control. F: Western blot analysis of the EPO receptor. EPOR expression was observed in AR42J cell lysate using an anti-EPOR antibody as described in MATERIALS AND METHODS. Lane marked as kDa represents the Precision Plus protein marker Western C standard (Bio-Rad). Lane 1, positive control (lysate from human leukemia cell line, K562). Lane 2, AR42J cell lysate. Lane 3, Western blot analysis of the AR42J cells after transfection with negative control for EPOR siRNA. Lane 4, Western blot analysis of the AR42J cells after transfection with siRNA for EPOR. Reduction in ~66-kDa band was observed (between lane 2 and 4). Lane 5, Western blot analysis with AR42J cell lysate treated with only secondary antibody (1:2,000) as a negative control. Lane 6, additional negative control. AR42J cell lysate of same amount (25 µg) was used for blotting with preincubated EPOR anti body as described in MATERIALS AND METHODS. Western blotted membranes were stripped and reprobed with β-actin antibody for the demonstration of equal loading.

 
Effect of EPO on DNA synthesis of AR42J cells. After ascertaining the presence of EPOR on AR42J cells, we initiated experiments to find the functional significance of EPOR expressed by these cells by incubating them with EPO. In an initial study using the colorimetric BrdU ELISA kit, we determined that 5 mU/ml EPO increased BrdU incorporation into DNA at 48 h compared with the control without EPO. We then undertook a detailed investigation regarding the proliferative pattern of AR42J cells with and without 5 mU/ml EPO over a period from 24 to 96 h. Incubation of cells with 5 mU/ml of EPO induced DNA synthesis, and the level was significantly higher compared with cultures without it (P < 0.05, Fig. 2A). Maximum enhancement of proliferation was seen at 48 h, and it was 3.05-fold above control. At 72 and 96 h, BrdU incorporation decreased significantly from the 48-h values in both, with, and without EPO cultures. However, the difference between EPO added and control cultures were still maintained at these intervals as well (Fig. 2A).


Figure 2
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  		Fig. 2. Enhancement of proliferation of AR42J cells by EPO. A: AR42J cell proliferation by EPO. AR42J cells were plated in 96-well culture plates, serum-starved overnight, and then treated with 5 mU/ml of EPO in F-12 medium containing 10% serum for 24 to 96 h. Control cells had medium containing 10% serum alone for the same length of time. Four hours before the final incubation time, 10 µM bromodeoxyuridine (BrdU) was added to the medium and incorporation of BrdU was measured as described in MATERIALS AND METHODS. Maximum BrdU incorporation was seen at 48 h in EPO-added samples. Data shown are means ± SE of 6 separate experiments. B: EPO dose response on the proliferation of AR42J cells. AR42J cells were plated as described above and treated with various concentrations of EPO (from 1 to 10 mU/ml). Proliferation was analyzed by BrdU incorporation assay at 48-h intervals as described earlier. Maximum BrdU incorporation was seen with 5 mU/ml of EPO among the doses used. Data shown are means ± SE of 6 separate experiments (*P < 0.05).

 
To determine the concentration of EPO that induced maximum proliferation, AR42J cells were incubated for 48 h with different doses of EPO, from 1 to 10 mU/ml. With all of the doses of EPO used, proliferation of AR42J cells was elevated above the control (without EPO). The maximum elevation in proliferation occurred with 5 mU/ml EPO, with a proliferation equivalent of 1.87-fold above the control cultures (P < 0.05) (Fig. 2B). We used this dose (5 mU/ml) of EPO for all our subsequent studies.

As described previously, we observed the presence of EPOR on AR42J cell surfaces, and to relate this finding with the proliferation of AR42J cells, we performed experiments using rabbit polyclonal anti-EPOR antibody H-194 (sodium azide free). Proliferation in this case was measured by the WST-8 cell-counting kit at 48 h of incubation with or without 5 mU/ml of EPO. When cells were incubated without EPO the proliferation was 1.06 ± 0.01 arbitrary units, which increased by 1.7-fold to 1.82 ± 0.01 arbitrary units when 5 mU/ml EPO was also present in the medium. With the pretreatment of AR42J cells with 0.02 µg/ml of H-194 anti-EPOR antibody alone, the proliferation of these cells was similar to the control, and the value was 1.02 ± 0.02 arbitrary units. When AR42J cells were pretreated with a similar quantity of anti-EPOR and then incubated with EPO, the enhancement of AR42J cells proliferation, as seen with EPO addition, was completely suppressed, the value being 1.16 ± 0.02 arbitrary units, suggesting that EPOR plays a significant role in EPO-enhanced proliferation of AR42J cells.

EPO activates ERK and JNK in AR42J cells. The next series of studies were conducted to elucidate EPO-mediated activation of three MAPK signals in AR42J cells; namely, ERK1/2, JNK1/2, and p38 MAPK, and these were performed by Western blot analysis. Figure 3, A and C, illustrates the phosphorylation of ERK1/2 by EPO in AR42J cells. The process was rapid and sustained with a maximum activation in phosphorylation occurring at 5 min compared with the control (increase by 3.06-fold). However, the amount of total ERK1/2 protein was not affected by EPO treatment at any time point tested (Fig. 3A, top blot).


Figure 3
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  		Fig. 3. Activation of extracellular regulatory kinase 1/2 (ERK1/2) and c-Jun NH2 terminal kinase 1/2 (JNK1/2) but not p38 when treated by EPO. A, B, and E: representative Western blots of total and phosphorylated ERK1/2, JNK1/2, and p38 in EPO-treated AR42J cells; C and D: analysis of band intensities of phospho-ERK1/2 and phospho-JNK1/2, respectively. Cells were treated with 5 mU/ml of EPO for the indicated time (from 5 min to 4 h) or left untreated (control). Whole cell lysates were prepared and equal amounts of protein (25 µg for pERK-1/2 and 50 µg for pJNK and p38) were loaded on to 4–12% bis-Tris NuPAGE gel, and Western blot analysis was performed using specific rabbit polyclonal antibodies (anti-phospho ERK1/2, JNK1/2, and p38). The same blots were stripped and reprobed with their respective total anti-mitogen-activated protein kinase (MAPK) antibodies. Blots show that there was no effect on the quantity of total ERK1/2, JNK1/2, and p38, and indicated equal amounts of protein loading in each lane. Plots were derived from four separate experiments and means ± SE of band intensities in fold induction above control of phospho ERK1/2 (C) and phospho JNK1/2 (D) were plotted. In both C and D, zero on the x-axis represents the controls (*P < 0.05, compared with control).

 
We then examined the activation of JNK1/2 by EPO in AR42J cells. Immunoblotting the cell lysates with specific antibody to phospho-JNK1/2 showed that similar to ERK1/2, JNK1/2 was also stimulated with EPO, although the time course of JNK1/2 activation was markedly different from that of ERK1/2. JNK1/2 phosphorylation was stimulated maximally at 30-min intervals (1.8-fold; Fig. 3D). In contrast, EPO did not activate steady-state levels of total JNK1/2 at any time point examined (Fig. 3B, top blot). We observed a very clear biphasic activation of ERK1/2 and JNK1/2 after EPO treatment. In both cases small, although statistically nonsignificant, second peaks of phosphorylated ERK1/2 and JNK1/2 were seen at the 3-h interval (Fig. 3, C and D). EPO had no effect on the phosphorylation of p38 MAPK at any time point tested (Fig. 3E), despite the fact that there were reasonably abundant amounts of p38 proteins present in the total cell lysates of EPO-treated and untreated AR42J cells.

In our MAPK studies we found that ERK1/2 and JNK1/2 were induced maximally at 5 and 30 min, respectively. We next examined the subcellular distribution of phosphorylated ERK1/2 and JNK1/2 by immunostaining. AR42J cells were stimulated with 5 mU/ml of EPO for 5 and 30 min for ERK1/2 and JNK1/2, respectively, as described previously. As shown in Fig. 4, B and D, pERK1/2 and pJNK1/2 fluorescence were significantly higher after 5 and 30 min of EPO treatment, respectively, compared with untreated cells (Fig. 4, A and C).


Figure 4
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  		Fig. 4. Immunofluorescence study of AR42J cells for ERK1/2 and JNK1/2 induction. A–D: AR42J cells were grown and treated with 5 mU/ml of EPO for 5 min for ERK1/2, or 30 min for JNK-1/2, or left untreated as controls for the same time periods. Cells were immunolabeled for phospho-ERK1/2 and phospho-JNK1/2 with respective antibodies, followed by anti-rabbit FITC antibody treatment, and then observed under the fluorescent microscope. A and B show phosphorylations of ERK1/2 in untreated controls and EPO-treated samples, whereas C and D show corresponding phosphorylations of JNK1/2. The increase in fluorescence and subcellular localization can be seen in EPO-treated samples (B and D).

 
Kinetics of EPO-stimulated expression of Elk-1 and c-Jun in AR42J cells. Since we observed that EPO activates ERK1/2 and JNK1/2 in AR42J cells, it was of interest to determine the functional activities of ERK1/2 and JNK1/2 in these cells after EPO treatment. Cell lysates were prepared from EPO-treated and untreated samples. Figure 5C shows the activity of immunoprecipitated ERK1/2, as determined by Elk-1 phosphorylation. ERK1/2 activity significantly increased fourfold at 5 min, gradually decreased with time, and attained its lowest activity at 4 h but still higher than the control cells without EPO (P < 0.05, Fig. 5C).


Figure 5
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  		Fig. 5. EPO-induced ERK and JNK kinase activities in vitro. A–D: kinase activities. ERK and JNK kinase activities were measured by a nonradioactive kinase assay kit. Cell extracts of AR42J cells treated with 5 mU/ml EPO for 5 min to 4 h (similar to Fig. 2) were utilized for the ERK and JNK kinase assays. Activities were measured by employing recombinant GST-Elk-1 (for ERK1/2) and GST-c-Jun (for JNK1/2) as substrates. Phosphorylated Elk-1 and c-Jun were assessed by Western blot analysis using antiphospho-Elk-1 and antiphospho-c-Jun antibodies for ERK1/2 and JNK1/2 kinase, respectively, followed by autoradiography. Representative autoradiograms are shown in A for pElk-1 and B for pc-Jun. The plots in C and D show the fold induction of pElk-1 and pc-Jun compared with their respective controls. In both C and D, zero on the x-axis represents the controls. The means ± SE of 4 independent experiments were plotted. The amount of immunoprecipated ERK1/2 and JNK1/2 were determined by reprobing the membranes with total anti-Elk-1 and total anti-c-Jun antibodies, and representative blots were included in A and B (*P < 0.05.)

 
Next, we examined the protein kinase activity of JNK1/2 at a various time points after EPO stimulation. As depicted in Fig. 5D, JNK1/2 activities were elevated upon EPO treatment compared with JNK1/2 activities seen in nonstimulated cells. A marked increase in the activities was detected within 15 min and lasted up to 1 h after EPO treatment, returning to normal levels by 4 h (Fig. 5D). However, the level of phospho-JNK1/2 reached its maximum at 30 min, whereas JNK1/2 activity measured by kinase assay reached a maximum at 15 min (Fig. 5D). The kinetics of EPO-induced Elk-1 (Fig. 5C) was parallel to the kinetics of ERK1/2 activation to a very close degree, whereas for JNK1/2, maximum phosphorylation and kinase activity did not show a parallel trend. Both ERK1/2 and JNK1/2 kinase activity did not show any biphasic activation like ERK1/2 and JNK1/2 phosphorylation (Fig. 3, C and D). Immunoblot analysis of the same membranes showed that the amounts of immunoprecipitated ERK1/2 and JNK1/2 were similar (Fig. 5, A and B, upper blots, respectively).

MAPK, ERK-1/2, and JNK-1/2 are involved in EPO-induced AR42J cell proliferation. Since ERK and JNK were activated in AR42J cells by EPO, we sought to determine the role of ERK and JNK in EPO-enhanced cell growth and proliferation of these cells using the specific MAPK inhibitors PD98059 (50 µM, for ERK1/2) and SP600125 (15 µM, for JNK1/2). The dose of each inhibitor was determined using Western blot analyses that were required to significantly inhibit EPO-induced EKR1/2 and JNK1/2 in these cells at 5 and 30 min, respectively (Fig. 6, C and D). Cell proliferation must proceed by the increase in DNA synthesis, and, therefore, the capability of PD98059 and SP600125 to inhibit the EPO-dependent incorporation of BrdU into DNA was first examined. As shown in Fig. 6A, DNA synthesis at 48 h increased 2.3-fold in EPO-added cultures, and this was inhibited up to the control level when cells were pretreated with 50 µM of PD98059 or 15 µM of SP600125, respectively (P < 0.05). We also observed a similar reduction in the proliferation at 48 h using the WST-8 cell-counting kit (P < 0.05, Fig. 6B). Addition of these inhibitors alone to cultures did not result in any decrease in cell count below that of the control (Fig. 6, A and B), ruling out the toxicity of these inhibitors on cell proliferation in a normal setting.


Figure 6
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  		Fig. 6. Effect of mitogen-activated protein/ERK kinase (MEK) (ERK) and JNK inhibitors on EPO-enhanced proliferation of AR42J cells. A and B: AR42J cells were cultured as previously described and treated with 50 µM of MEK inhibitor (PD98059) or 15 µM of JNK inhibitor (SP600125) for 1 h. Control cells had the equivalent volume of DMSO. Half of these samples were further treated with 5 mU/ml of EPO. The cell proliferation of all the samples was measured at 48 h by colorimetric-based assays using BrdU incorporation into AR42J cells (A) or WST-8 cell counting kit (B), as described in MATERIALS AND METHODS. Bars on the figure represent means ± SE of 4 separate experiments (*P < 0.05 between control and EPO added samples; **P < 0.05 between EPO and EPO + inhibitor added samples). C and D: effect of PD98059 (50 µM) and SP600125 (15 µM) on EPO-induced ERK1/2 and JNK1/2. AR42J cells were cultured and treated with EPO for 5 and 30 min, similar to the legend of Fig. 2. For inhibitor studies, cells were pretreated with 50 µM of PD98059 or 15 µM of SP600125 and incubated for 1 h before the addition of EPO. Western blot analysis was performed, and membranes were probed with antiphospho-ERK1/2 or antiphospho-JNK1/2. PD98059 blocked the EPO-induced ERK1/2 and SP600125 blocked JNK1/2. Membranes were stripped and reprobed with total anti-ERK1/2 and total anti-JNK1/2. Both inhibitors had no effect on total ERK1/2 and JNK1/2, indicating equal protein loading. Blots are representative of 4 separate studies.

 
Cell cycle progression and S-phase entry in EPO-treated AR42J cells. To further verify the proliferative effect of EPO on AR42J cells, cell-cycle analysis was performed by flow cytometry. Figure 7A shows that 71.6 ± 1.0% cells were in the G0/G1 phase at 0 h, with 9.2 ± 0.6% and 12.8 ± 0.6% at the S and G2/M phases, respectively. After the culture of these cells with 5 mU/ml EPO for 48 h, the percentage of cells in the DNA synthesis phase (S phase) was increased significantly to 33.5 ± 0.5% (P < 0.05), accompanied by a corresponding decrease in the G0/G1 phase of the cell cycle (38.5 ± 0.9%) (Fig. 7D). In cultures without EPO, cells in the S phase at that interval were significantly less, 15.6 ± 0.5%, with 55.3 ± 0.9% remaining at the G0/G1 phase (Fig. 7C). At 72 and 96 h there were no marked differences in the cell-cycle distribution at any phase (data not shown). Thus the observed cell-cycle distribution confirmed our results with BrdU incorporation and WST-8 cell counting kit. These results suggest that EPO enhanced the proliferation of AR42J cells by inducing cell-cycle progression.


Figure 7
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  		Fig. 7. EPO-enhanced cell cycle progression is mediated through ERK and JNK. AR42J cells were plated in 24-well plates, serum starved for 24 h, and then cultured in 10% FBS medium. Asynchronous cells were cultured with 5 mU/ml of EPO alone or in the presence of inhibitors, 50 µM of PD98059, or 15 µM of SP600125 for 48 h. Cells were fixed in 70% ethanol, DNA stained with propidium iodide, and subjected to flow cytometry analysis using the Guava EasyCyte mini system, for cell cycle phase distribution. Regions 1, 2, and 3 on each panel represent G0/G1, S, and G2/M phase of the cell cycle, respectively. Values plotted on the panels are means ± SE of 4 independent studies.

 
We utilized the inhibitors of ERK (PD98059) and JNK (SP600125) to analyze these results further and examined the percentage of cells distributed in different phases of the cell cycle after treatment with these inhibitors, using flow cytometric analyses previously described. Both SP600125 and PD98059 abolished the increase in S phase cells 48 h after EPO treatment, and the values were 13.1 ± 0.2% and 11.8 ± 0.2%, respectively, compared with the value of 33.5 ± 0.2% in EPO-alone added cultures (Fig. 7, E and F). Thus activation of both ERK and JNK appears to be critical for the EPO-mediated growth and proliferation of AR42J cells.

Inhibition of EPO-induced proliferation of AR42J cells by JNK-1/2 RNA interference. Previous results demonstrate that ERK and JNK are both required for EPO-mediated proliferation of AR42J cells; inhibitor studies also show that inhibition of either ERK or JNK can reduce EPO-dependent proliferation of these cells. To confirm this further, we employed the siRNA approach in conjunction with inhibition of JNK1/2, which should determine the significance of JNK1/2 for the EPO-enhanced proliferation of AR42J cells. The specific knock down of JNK1/2 protein levels was investigated by Western blot analysis. As shown in Fig. 8C, small interfering JNK (siJNK) transfection effectively decreased the total JNK1 by ~52% and total JNK2 ~97%. Transfection efficiency, using fluorescent oligo as described in MATERIALS AND METHODS was estimated to be 60–70%. Reduction in JNK mRNA levels as observed by real-time PCR was ~60% in siJNK-transfected cells (data not shown). Thus reduction in JNK protein was consistent with transfection efficiency and decreased mRNA levels. Treatment of siJNK suppressed EPO-enhanced proliferation of AR42J cells significantly compared with the untransfected cells treated with EPO, indicated by both WST-8 absorption and BrdU incorporation methods (Fig. 8, A and B). Reduction in proliferation was coincided with decreased level of phosphorylated JNK1/2 in siJNK-treated cells (Fig. 8D), suggesting that depletion in JNK1/2 protein by JNK siRNA inhibited the EPO-enhanced proliferation of AR42J cells by inhibiting JNK phosphorylation. Nonspecific control siRNA, used as a negative control, had no effect on total JNK proteins (Fig. 8C). Although the phosphorylated levels of JNK1/2 were increased, the increases were not similar to EPO-only-treated cells (Fig. 8D). Proliferation of nonspecific control siRNA-transfected cells were significantly increased above control, as observed in both CCK8 or BrdU methods (Fig. 8, A and B). Experiments repeated several times with control siRNA along with JNK stealth siRNA showed similar observations. At this point it was not clear why nonspecific control siRNA transfection was showing the increase in WST-8 absorbance and BrdU incorporation in AR42J cells with or without EPO. We had to assume that the nonspecific control siRNA might have knocked down some off-target genes, and alternatively other pathways were triggered, resulting in an enhanced proliferation of AR42J cells by the induction of cellular metabolic activity and DNA synthesis.


Figure 8
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  		Fig. 8. Effect of JNK siRNA on EPO-enhanced proliferation of AR42J cells. A and B: AR42J cells were transfected with 25 nM of JNK1/2 stealth siRNA or nonspecific control siRNA (negative control) as described under MATERIALS AND METHODS. After transfection, cells were washed with serum-free medium and cultured in serum-containing medium. Cultures were treated with 5 mU/ml of EPO for 48 h, and proliferation was evaluated using WST-8 cell counting (A) as well as BrdU incorporation method (B). Values presented are means ± SE of 3 separate experiments (*P < 0.05 between control and EPO added samples, **P < 0.05 between control and siRNA transfected samples with EPO addition). C: after siRNA transfection, expression of total and phospho-JNK1/2 protein was determined by Western blot analysis. This shows an efficient inhibition of total JNK-1/2 protein levels by JNK siRNA transfection, whereas nonspecific control siRNA had no effect on total JNK1/2. The histogram shows the inhibition of total JNK1 and 2 from control values after siJNK transfection as calculated from 3 separate experiments. D. treatment of siJNK inhibited the EPO-induced phospho-JNK1/2. Blot shown is the representative of three independent experiments. Same blots were stripped and reprobed for β-actin to confirm equal loading.

 

   DISCUSSION
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In the current study, we have demonstrated the expression of EPOR in AR42J cells by immunofluorescence, RT-PCR, and Western blot analysis studies. There are several antibodies available for the detection of EPOR. Whereas Elliott et al. (18) have examined some of these and found to detect different sizes of EPOR bands, Kirkeby et al. (37) have found most of them, including H-194 antibody, to detect a 66-kDa protein band. We have used H-194 antibody from Santa Cruz throughout our study. Our data shows that 5 mU/ml EPO stimulates proliferation of AR42J cells. The EPO dose used for this study was established after the dose-response study, and 5 mU/ml EPO was found to be optimum for this enhancement in proliferation. In earlier reports, EPO doses from 10 mU/ml to 200 U/ml were used in in vitro studies with different cell systems (3, 22, 26, 54, 62), which are higher than the dose of 5 mU/ml we employed for AR42J cells. Feldman et al. (21) have shown the proliferation of four out of five prostate cancer cell lines at 1 U/ml. Cell specificity may be the reason for this difference. Our observation of EPO-enhanced proliferation of AR42J cells raises an important question, whether activation of the MAPK cascade is involved in this process. To analyze this in detail, we employed a variety of separate but complementary experiments.

Our time-course evaluation of the EPO effect on ERK, as well as JNK activation, showed that ERK1/2 is activated to a maximum extent within 5 min, but JNK1/2 is maximally activated after 30 min. The magnitude and duration of these MAPK activities are very critical for cell-signaling decisions, as transient activation of MAPK triggers proliferation, whereas sustained activation allows differentiation, as shown previously using other cell systems (11, 45, 52). The mechanism of activation of MAPK by EPOR in tumor cells is elusive, but it is likely that the carboxyl-terminal region of the EPOR plays important role in the activation of MAPK and it might occur depending upon either Ras-Raf-1 or PKC activation, as shown previously in human erythroleukemia and EPO-dependent megakaryoblastic cell lines (8, 38, 63, 67). It might also occur by a Ras-independent mechanism as shown in BaF3 cells (9). Whereas it has been well documented that ERK, activated by various stimuli, including growth factors, is implicated in proliferation, and JNK is involved in stress-induced apoptosis (5, 7, 41, 42, 44, 55, 58), an accumulating body of evidence has recently suggested that JNK plays a key role in cell survival and proliferation for a variety of cell types (16, 31, 32, 44, 53). Other studies have also suggested that c-Jun might have a pro-apoptotic or prosurvival role depending on the specific context (20, 22).

Using specific kinase inhibitors, we have shown that activation of the MAPK pathway is involved in EPO-enhanced AR42J cell proliferation. Pretreatment with MEK inhibitor PD98059 or JNK inhibitor SP600125 attenuated EPO-enhanced cell proliferation and the cell cycle transition. More importantly JNK, together with ERK activation is required for EPO-enhanced AR42J cell proliferation, and this is clearly shown by inhibiting JNK or ERK using pharmacological inhibitors. The specificity of MAPK inhibition was further confirmed on the molecular basis by employing siRNA for JNK. It has also been shown that EPO-induced proliferation of HDC57 cells and other cell lines are blocked by the JNK inhibitor SP600125 (16, 28, 31), suggesting an important role of JNK in proliferation. Inhibition of basal JNK activity by SP600125 or antisense oligonucleotides has attenuated cell proliferation and cell cycle progression in the KB-3 human carcinoma cell line (16). In addition, both overexpression of a dominant negative JNK mutant and pharmacological inhibition of JNK has blocked the platelet-derived growth factor-induced proliferation in smooth muscle cells (33, 72). Our result with siRNA for JNK has also shown clearly that EPO-enhanced proliferation is inhibited, as indicated by BrdU incorporation, as well as WST-8 cell counting kit. Involvement of JNK with cell growth as shown in our study is consistent with several previous reports in different cell types using various stimuli, including EPO (16, 20, 23, 31, 36, 44, 53).

The decrease in proliferation observed with inhibitors could be due to growth arrest, so we analyzed cell growth with propidium iodide staining and flow cytometry. Our results demonstrated a shift of cells in the S phase after EPO treatment and an accumulation of cells in the G0/G1 phase in the presence of PD98059 or SP00125. Cells treated with PD98059 or SP600125 have also been shown to cause G0/G1 and G2/M arrest in a number of cell lines (10, 16, 43, 47, 61, 69). Our data, therefore, demonstrate that growth inhibition of EPO-treated cells by PD98059 and SP600125 was caused primarily by arrest at the G0/G1 phase of cell cycle, as evidenced by an accumulation of cells in the G0/G1 phase and reduction in growth rate, as a lesser number of cells were in the DNA synthesis phase (S phase). c-Jun and Elk-1 have long been demonstrated to be necessary for a G1 to S-phase progression (59, 61). To demonstrate directly that EPO-induced ERK and JNK kinases are active and capable of activating Elk-1 and c-Jun, which are involved in the induction of growth-related genes, we studied the phosphorylation of Elk-1 and c-Jun. As shown in Fig. 5, phosphorylation of Elk-1 and c-Jun correspond to the kinetic profiles of the EPO-induced activation of ERK and JNK (Fig. 3). Based on our observations, we can conclude that ERK1/2 and JNK1/2 are clear positive regulators of intracellular pathways leading to EPO-enhanced proliferation of AR42J cells through phosphorylation and activation of such growth-related factors. This observation is in contradiction to our earlier results of activation of only ERK1/2 by nicotine in AR42J cells to enhance the proliferation without the activation of JNK1/2 (5). These two results indicate that different stimuli act in a different manner in the same cell type to enhance the proliferation. The evidence presented here strongly supports the conclusion that inhibition of cell proliferation is a direct consequence of either ERK or JNK inhibition. However, it is not clear whether EPO-enhanced proliferation of AR42J cells, which is clearly mediated through the joint activation of ERK and JNK, is dependent on activation of cytosolic JAK2/STAT. STAT5 is a cell survival signal, and according to Kirkeby et al. (37), anti-EPOR antibody H-194 has not been able to suppress EPO-induced STAT5 signaling in UT-7 cells. In the AR42J cell system, our preliminary data indicates that EPO-induced STAT5 is suppressed by H-194 (data not given). The discrepancy between our results and those of Kirkeby et al. (37) may be due to the differences in the two cell systems. Because multiple signal transduction pathways can interact to regulate proliferation and cell cycles, our ongoing studies are looking into the role of JAK/STAT in EPO-induced ERK/JNK signaling in AR42J cells to investigate this aspect further.

The significant finding of our study is the observation that EPO-enhanced AR42J cell proliferation is ERK dependent, as well as JNK dependent. To the best of our knowledge, our study is the first to report this phenomenon in tumor cells. Our conclusion of involvement of both MAPKs in tumor cell line proliferation is also supported by reports of involvement of ERK and JNK in the proliferation of nontumorous EPO-dependent erythroid cells (30), as well as in platelet-derived growth factor-induced proliferation of oesteoblastic MC3T3-E1 cells (46). In summary, our present observation shows that AR42J cells express EPOR. EPO-stimulated proliferation of AR42J cells indicates that the EPORs expressed by these cells are functional, and that EPO receptor-meditated stimulation is associated with activation of ERK and JNK pathways. Furthermore, our study suggests that activation of either ERK or JNK alone is insufficient to stimulate EPO-mediated cell proliferation in these cells. Instead, ERK and JNK both play a crucial role for EPO-enhanced proliferation and cell cycle progression of AR42J cells. With the selective suppression of one of these components of MAPK with specific inhibitors, the possibility appears to identify therapeutic targets within the signaling pathway of EPOR-bearing tumor cells.


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This work was partially supported by funds from the Central Arkansas Veterans Healthcare System.


   ACKNOWLEDGMENTS
 
We thank Drs. Peter M. Price, Judith Megyesi, and Piotr Zimniak of the University of Arkansas for Medical Sciences for valuable suggestions and discussion during this work, and Dr. Sharda P. Singh for performing RT-PCR.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. B. Udupa, Medical Research, Central Arkansas Veterans Healthcare System, 4300 West Seventh St., Little Rock, AR 72205-5446 (e-mail: udupakodetthoor{at}uams.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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