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Clinical Cancer Research Vol. 12, 49-56, January 2006
© 2006 American Association for Cancer Research

Human Cancer Biology

PC Cell–Derived Growth Factor Confers Resistance to Dexamethasone and Promotes Tumorigenesis in Human Multiple Myeloma

Wengang Wang1, Jun Hayashi1 and Ginette Serrero2,3

Authors' Affiliations: 1 Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy; 2 Program in Oncology, Marlene and Stewart Greenebaum Cancer Center of the University of Maryland, Baltimore, Maryland; and 3 Research and Development Department, A&G Pharmaceutical, Inc., Columbia, Maryland

Requests for reprints: Ginette Serrero, A&G Pharmaceutical, Inc., 9130 Red Branch Road, Suite U, Columbia MD 21045. Phone: 410-884-4100; Fax: 410-884-1607; E-mail: gserrero{at}agrx.net.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: We have shown previously that the 88 kDa glycoprotein PC cell–derived growth factor (PCDGF/GP88) is expressed and acts as an autocrine growth factor in human multiple myeloma cells. The present study investigates whether PCDGF/GP88 expression in multiple myeloma cells leads to the development of resistance to dexamethasone, a conventional drug for multiple myeloma patients.

Experimental Design: PCDGF functions and signaling pathways in dexamethasone-induced apoptosis were studied using a representative dexamethasone-sensitive multiple myeloma cell line ARP-1. The effect of PCDGF/GP88 was further confirmed in PCDGF/GP88–overexpressed ARP-1 cells.

Results: Dexamethasone inhibits cell growth and induces apoptosis in a time- and dose-dependent fashion. Exogenous addition of PCDGF/GP88 to the ARP-1 cells prevented dexamethasone-induced apoptosis as examined by flow cytometry analysis and poly(ADP-ribose)polymerase cleavage assay. Signaling studies showed that mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and nuclear factor-{kappa}B were involved in the antiapoptotic effect of PCDGF/GP88. Overexpression of PCDGF/GP88 in ARP-1 cells rendered the cells refractory to dexamethasone-mediated apoptosis, enhanced their ability to form colonies in soft agar, and to form tumors in vivo without any change in glucocorticoid receptor expression and function.

Conclusion: These data suggest that expression of PCDGF/GP88 confers resistance to dexamethasone and increase tumorigenesis of multiple myeloma cells in mouse xenografts. Our data here also raises the possibility of PCDGF/GP88 as a potential therapeutic target for dexamethasone-resistant multiple myeloma.

Multiple myeloma is a clonal plasma cell neoplasm that leads to the accumulation of malignant plasma cells (1). There are ~14,400 new multiple myeloma cases and 11,200 deaths from multiple myeloma annually in the United States (2). The glucocorticoid analogue dexamethasone is frequently used in the treatment of multiple myeloma, alone or in combination with cytotoxic drugs, because of its ability to induce apoptosis of multiple myeloma cells (3). Dexamethasone-induced apoptosis is associated with a significant decrease in mitogen-activated protein kinase (MAPK) and p70 S6 kinase (p70S6K) activities. In addition, dexamethasone-induced apoptosis was shown to be associated with decreased nuclear factor-{kappa}B (NF-{kappa}B) DNA binding and {kappa}B-dependent transcription (4). However, the majority of multiple myeloma patients who initially respond to the treatment of dexamethasone usually relapse within 2 years (3, 5).

Acquired glucocorticoid resistance may result from mutations or defective splicing of the glucocorticoid receptor (6). The development of resistance to dexamethasone may also be due to the overexpression of growth factors. High expression levels of interleukin-6 correlates with the resistance to dexamethasone in multiple myeloma cell clones obtained from the bone marrow of patients with multiple myeloma at different clinical stages (7).

PC cell–derived growth factor (PCDGF), also called progranulin, is an 88 kDa glycoprotein comprising a 68 kDa protein core and a 20 kDa carbohydrate moiety (PCDGF/GP88; ref. 8). It was originally isolated as an autocrine growth factor from the culture medium of the highly tumorigenic mouse teratoma cell line PC. PCDGF/GP88 is a growth modulator for a variety of cell lines of mesenchymal and epithelial origin (9, 10). Recently, our laboratory has shown that PCDGF/GP88 was highly expressed in human multiple myeloma cell lines. A higher level of PCDGF/GP88 was expressed in dexamethasone-resistant multiple myeloma cells compared with dexamethasone-sensitive cells (11). PCDGF/GP88 acted as an autocrine growth factor and stimulated MAPK as well as phosphatidylinositol 3-kinase (PI3K) but not the Janus tyrosine kinase-signal transducer and activator of transcription pathways (11). Pathologic studies indicated that PCDGF/GP88 was expressed in immunoglobulin light chain expressing multiple myeloma cells in the bone marrow smears of multiple myeloma patients (11).

Because dexamethasone plays an important role in the treatment of multiple myeloma and because the overexpression of PCDGF/GP88 is associated with tamoxifen resistance in breast cancer cells (12), the present study examined whether PCDGF/GP88 exerted similar effects on dexamethasone-induced apoptosis in human multiple myeloma. The ARP-1 cell line was used in this present study because it is a representative dexamethasone-sensitive multiple myeloma cell line to investigate effects of growth factors on dexamethasone-induced apoptosis (13).


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell culture. Human multiple myeloma cell line ARP-1, originally established at the Arkansas Cancer Research Center from bone marrow aspirates of multiple myeloma patients (13), was a gift from Dr. Robert G. Fenton (School of Medicine, University of Maryland, Baltimore, MD). RPMI 1640 and fetal bovine serum (FBS) were obtained from Invitrogen Life Technologies (Carlsbad, CA). ARP-1 cells were maintained in 10% charcoal-treated FBS–supplemented RPMI 1640.

Determination of PCDGF/GP88 mRNA and protein expressions. PCDGF/GP88 mRNA and protein expressions were determined by Northern blot analysis and immunoprecipitation followed by Western blot analysis, respectively, as described previously (14). 28S RNA was used as an internal control for Northern blot analyses. ß-actin detected by a polyclonal antibody (Novus Biologicals, Littleton, CO) was used as an internal control for Western blot analyses.

Determination of the cleavage of poly(ADP-ribose) polymerase. ARP-1 cells were seeded at a density of 4 x 104 cells/mL. Dexamethasone (10–7 mol/L) and/or 200 ng/mL PCDGF/GP88 were added for either 24 or 48 hours. ARP-1 cells were lysed in 0.1 mL lysis buffer [62.5 mmol/L Tris-HCl (pH 6.8), 6 mol/L urea, 10% glycerol, 2% SDS, 0.00125% bromophenol blue, and 5% ß-mercaptoethanol], sonicated for 15 seconds, and then boiled for 10 minutes. Poly(ADP-ribose)polymerase (PARP) was detected by Western blot analyses using 1 µg/mL monoclonal anti-PARP antibody (Oncogene, Boston, MA).

Flow cytometry analysis of ARP-1 cells treated with dexamethasone and PCDGF/GP88. After a 72-hour treatment with dexamethasone (10–7 mol/L) and/or 200 ng/mL PCDGF/GP88, 105 ARP-1 cells were collected, washed with PBS, and fixed in 70% cold ethanol at 4°C overnight. On the day of analysis, cells were treated with 0.1 mg/mL RNase (Stratagene, La Jolla, CA) for 1 hour at room temperature followed by a 30-minute incubation with 50 µg/mL propidium iodide (Sigma, St. Louis, MO). Cells were analyzed on a FACScan flow cytometer using the CELL Quest program (Becton Dickinson, Franklin Lakes, NJ). Apoptotic cells were detected as a hypodiploid population by ModFit LT program (VERITY software house, Topsham, ME).

Determination of MAPK and Akt phosphorylation. ARP-1 cells in culture were treated for 60 minutes with or without 30 µmol/L PD98059 before adding increasing concentrations of PCDGF/GP88. After 10-minute incubation, cells were lysed in Laemmli sample buffer. Lysate from 3 x 106 live cells was used for each sample. Phospho-extracellular signal-regulated kinase 1/2 (ERK1/2), total ERK2, phospho-Akt, and total Akt proteins were detected using anti-phospho-ERK1/2 (New England Biolabs, Beverly, MA), anti-ERK2 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-Akt, and anti-Akt antibodies (New England Biolabs), respectively.

Determination of NF-{kappa}B luciferase activity. The pNF-{kappa}B-Luc reporter plasmid (Clontech, Palo Alto, CA) and pSV-ß-Galactosidase (Promega, Madison, WI; used as internal control to determine transfection efficiency) were cotransfected in ARP-1 cells by electroporation. Transfected cells were plated in 12-well plates at 5 x 105 cells per well for 48 hours in the presence or absence of 10–7 mol/L dexamethasone and/or 200 ng/mL PCDGF/GP88. Each transfection was done in triplicate. Luciferase activities were determined with a luminometer and were calculated using ß-galactosidase activity to normalize for transfection efficiency. Luciferase activity values were provided as relative luciferase activity, corresponding to the ratio of luciferase activity of pNF-{kappa}B-Luc to pTAL-Luc.

Overexpression of PCDGF/GP88 in ARP-1 cells. ARP-1 cells were cultured at a density between 8 x 105 and 10 x 105 cells/mL. Cells were washed with PBS, resuspended in 0.4 mL Opti-MEM supplemented with 10% FBS, and transfected by electroporation with 5 µg plasmid DNA of pcDNA3 empty vector or pcDNA3 vector containing PCDGF/GP88 cDNA (pcDNA3-PCDGF/GP88; ref. 15). The cells were cultured in RPMI 1640 supplemented with 10% FBS. After 48-hour incubation, G418 (Invitrogen Life Technologies) was added to a final concentration of 0.4 mg/mL to select for transfected cells.

Glucocorticoid receptor functionality assay. ARP-1-EV or ARP-1-PCDGF/GP88 cells (5 x 105) were transfected with 1 µg pGRE-Luc and 1 µg pSV-ß-galactosidase or 1 µg pTAL-Luc and 1 µg pSV-ß-galactosidase by electroporation. Transfected cells were plated in 12-well plates at 5 x 105 per well for 48 hours in the presence or absence of 10–7 mol/L dexamethasone. Each transfection was done in triplicate. Luciferase activities were corrected using ß-galactosidase activity to normalize for transfection efficiency. Final luciferase activity was given as relative luciferase activity, which is the ratio of luciferase activity of pGRE-Luc to pTAL-Luc.

Colony-forming assay. Soft agar was prepared at concentrations of 1% and 0.6% just before use. The bottom soft agar layer (Difco, Detroit, MI) consisted of 0.5% agar in RPMI-10% charcoal treated FBS. The top agar layer (0.3% agar) contained 5,000 cells. For the dexamethasone-treated samples, dexamethasone was added in bottom and top layers at a final concentration of 10–7 mol/L. For other samples, the same volume of vehicle was added. After 3 weeks, the colonies were stained by 0.1% crystal violet (Fisher Scientific, Pittsburgh, PA) and counted.

In vivo tumorigenicity study. ARP-1-EV or ARP-1-PCDGF/GP88 cells (5 x 106 per site) were s.c. inoculated in into 6-week-old female athymic nude mice (National Cancer Institute, Frederick, MD). The appearance and size of the tumors were examined daily. The tumor volume was calculated as (W2 x L / 2), where W is the width and L is the length of tumors. Mice were euthanized 22 days after inoculation to measure tumor weights.

Statistics. Experiments were carried out in triplicate and repeated thrice. The result was expressed as mean ± SD. Two-tailed t test was used for significance testing of the data. P < 0.05 was taken as the level of significance.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Regulation of PCDGF/GP88 expression in ARP-1 cells by dexamethasone. We have previously reported that PCDGF/GP88 is an autocrine growth factor produced by multiple myeloma cell lines and multiple myeloma cells from patients (11). PCDGF/GP88 is produced by ARP-1 cells and stimulate their growth (11). Because it induces apoptosis of multiple myeloma cells, the effect of dexamethasone on PCDGF/GP88 expression was examined using dexamethasone-sensitive multiple myeloma cell line ARP-1. As shown in Fig. 1A, after a 24-hour treatment with dexamethasone (10–8, 10–7, 10–6, and 10–5 mol/L), PCDGF/GP88 mRNA expression was inhibited in a dose-dependent fashion (34%, 40%, 52%, and 64%, respectively) as measured by Northern blot analyses. Dexamethasone-mediated inhibition of PCDGF/GP88 mRNA expression reached a maximum level after 24 hours of dexamethasone treatment of cells. PCDGF/GP88 production by the cells was inhibited by 68%, 72 hours after the dexamethasone treatment (10–7 mol/L) of the cells (Fig. 1B).



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  		Fig. 1. Effect of dexamethasone on PCDGF/GP88 mRNA and protein expressions and effect of PCDGF/GP88 on dexamethasone-induced cell death. ARP-1 cells were seeded at 4 x 104/mL in RPMI 1640 supplemented with 10% charcoal-treated FBS and treated with different concentrations of dexamethasone. A, dose response of dexamethasone (Dex) effect on PCDGF/GP88 mRNA expression after 24-hour treatment as measured by Northern blot. Top, PCDGF/GP88 mRNA expression. Bottom, 28S rRNA expression to indicate equal RNA loading. B, effect of dexamethasone on PCDGF/GP88 protein expression. Western blot analysis was carried out to measure PCDGF/GP88 expression levels. Top, PCDGF/GP88 protein expression. Bottom, ß-actin protein expression to indicate equal loading. Con, control. C, after 48-hour treatment, effect of PCDGF/GP88 on dexamethasone-induced death as measured by live cell density. D, after 48-hour treatment, effect of PCDGF/GP88 on dexamethasone-induced cell death as measured by percentage survival. Columns, mean; bars, SD. *, P < 0.05, compared with 10–7 mol/L dexamethasone-treated sample (second column), t test.

 
PCDGF/GP88 prevents dexamethasone-mediated death of ARP-1 cells. Because PCDGF/GP88 is an autocrine growth factor for ARP-1 cells, the dexamethasone-mediated inhibition of PCDGF/GP88 production may be one of processes of apoptosis induced by dexamethasone. To examine if this reduction in PCDGF/GP88 production is important for the apoptotic effect of dexamethasone, ARP-1 cells were cultivated in the presence of 10–7 mol/L dexamethasone with or without PCDGF/GP88. As shown in Fig. 1C, 48 hours treatment of cells with dexamethasone resulted in a 54% inhibition of live cell density. Addition of PCDGF/GP88 at concentrations of 50 and 200 ng/mL in the presence of dexamethasone reduced the inhibition to 41% and 17%, respectively (Fig. 1C). Higher concentration of PCDGF/GP88 (400 ng/mL) did not have any additional effect. Similar results were found on cell viability (Fig. 1D). These results indicated that PCDGF/GP88 prevented dexamethasone-mediated apoptosis in ARP-1 cells.

PCDGF/GP88 inhibits dexamethasone-induced apoptosis as measured by PARP cleavage and flow cytometry. To further analyze the effect of PCDGF/GP88 on dexamethasone-mediated apoptosis, experiments were carried out to examine whether PCDGF/GP88 could prevent the cleavage of PARP induced by dexamethasone. As shown in Fig. 2A, after 24- and 48-hour treatment of ARP-1 cells with 10–7 mol/L dexamethasone, the cleaved form of PARP was detected. The generation of cleaved form of PARP was not detected in ARP-1 cells treated with 200 ng/mL PCDGF/GP88 in the presence of 10–7 mol/L dexamethasone for 24 hours. After 48-hour treatment of cells with dexamethasone in the presence of PCDGF/GP88, the cleaved form of PARP was detected; however, the level was significantly less than that detected in the absence of PCDGF/GP88.



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  		Fig. 2. PCDGF/GP88 inhibits dexamethasone-induced apoptosis as measured by PARP cleavage and flow cytometry. ARP-1 cells were seeded as described above. Dexamethasone (10–7 mol/L) and PCDGF/GP88 at different concentrations were added. A, cells were collected at 24 and 48 hours. Western blot analysis was carried out to measure the intact and cleaved PARP. B, cells were collected at 72 hours. Cells were fixed in 70% cold ethanol at 4°C and stained with 50 µg/mL propidium iodide. The apoptotic percentage was detected as a hypodiploid population. Columns, mean; bars, SD. *, P < 0.05, compared with 10–7 mol/L dexamethasone-treated sample (second column), t test.

 
To further investigate PCDGF/GP88 effect on dexamethasone-induced apoptosis, flow cytometric analysis was carried out to examine the profile of ARP-1 cells undergoing dexamethasone-mediated apoptosis in the presence or absence of PCDGF/GP88. ARP-1 cells were stained by propidium iodide and apoptotic cells were detected as a hypodiploid population as described in Materials and Methods. As shown in Fig. 2B, the percentage of apoptotic cells in control and PCDGF/GP88 treated ARP-1 cells was 6.7% and 4.2%, respectively. The proportion of apoptotic cells increased to 58.2% when cells were treated with 10–7 mol/L dexamethasone for 72 hours. Addition of 200 ng/mL of PCDGF/GP88 significantly reduced to 36.1% the proportion of apoptotic cells.

The signaling pathways involved in the antiapoptotic effect of PCDGF/GP88. PCDGF/GP88 has been shown to activate MAPK (ERK) and PI3K signaling pathways in human multiple myeloma (11). Experiments were carried out to examine whether the activation of these kinases by PCDGF/GP88 was important for the antiapoptotic effect of PCDGF/GP88. As shown in Fig. 3A, 200 ng/mL PCDGF/GP88 significantly stimulated the phosphorylation of ERK1 and ERK2. This phosphorylation was inhibited by 30 µmol/L PD98059, a MAP/ERK kinase inhibitor. The PARP cleavage assay was concurrently measured. As shown in Fig. 3B, treatment of cells with 10–7 mol/L dexamethasone for 48 hours induced PARP cleavage (41% of PARP in the cleaved form as measured by a densitometer). The addition of PCDGF/GP88 (200 ng/mL) significantly reduced dexamethasone-mediated PARP cleavage (from 41% to 25%). The protective effect of PCDGF/GP88 on the dexamethasone-induced apoptosis was decreased when 30 µmol/L PD98059 was added to the culture. The treatment of cells with 30 µmol/L of PD98059 alone had no effect on PARP cleavage. Similar results were obtained when viable cell number was counted (Fig. 3C) and when cell viability was measured (Fig. 3D). These data show that MAPK pathway is involved in the antiapoptotic effect of PCDGF/GP88.



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  		Fig. 3. MAPK is involved in antiapoptotic effect of PCDGF/GP88. ARP-1 cells were seeded as described above. Dexamethasone (10–7 mol/L), 200 ng/mL PCDGF/GP88, and 30 µmol/L PD98059 were added. Cells were collected after 48-hour treatment. A, Western blot analysis of phosphorylated ERK1/2 and total ERK2 (as an internal control for equal loading). B, Western blot analysis of PARP cleavage. C, live cell density of ARP-1 cells after 48-hour treatment. D, the percentage survival of ARP-1 cells after 48-hour treatment. Columns, mean; bars, SD. P, PCDGF/GP88; PD, PD98059. *, P < 0.05 compared with control (first column), t test.

 
PI3K-Akt signaling pathway is also activated by PCDGF/GP88 as shown in Fig. 4A. PCDGF/GP88 (200 ng/mL) stimulated the phosphorylation of Akt in ARP-1 cells. This PCDGF/GP88–mediated Akt phosphorylation was inhibited by a PI3K inhibitor wortmannin at 50 nmol/L. Treatment of ARP-1 cells with 10–7 mol/L dexamethasone for 48 hours induced the PARP cleavage as shown above. The dexamethasone-induced PARP cleavage was significantly prevented by the addition of PCDGF/GP88 as previously shown. However, in the presence of 50 nmol/L of wortmannin, the preventive effect of PCDGF/GP88 on dexamethasone-mediated induction of PARP cleavage was inhibited (Fig. 4B). Similar results were obtained from cell growth assay where wortmannin inhibited PCDGF/GP88–mediated protective effect against dexamethasone-mediated growth inhibition (Fig. 4C and D). Our findings here show that PI3K-Akt pathway is involved for antiapoptotic effect of PCDGF/GP88.



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  		Fig. 4. PI3K is involved in antiapoptotic effect of PCDGF/GP88. ARP-1 cells were treated with 10–7 mol/L dexamethasone, 200 ng/mL PCDGF/GP88, and 50 nmol/L wortmannin were added. Cells were collected after 48-hour treatment. A, Western blot analysis result of phosphorylated Akt and total Akt expression (as an internal control for equal loading). B, Western blot analysis of PARP cleavage. C, live cell density of ARP-1 after 48-hour treatment. D, percentage survival of ARP-1 cells after 48-hour treatment. Columns, mean; bars, SD. *, P < 0.05 when compared with control (first column).

 
It was reported that dexamethasone-induced apoptosis is associated with decreased NF-{kappa}B DNA binding and {kappa}B-dependent transcription in ARP-1 cells (4). Because PCDGF/GP88 prevents dexamethasone-induced apoptosis, experiments were carried out to examine whether PCDGF/GP88 could prevent the dexamethasone-induced down-regulation of NF-{kappa}B. To test this, a reporter gene construct, pNF-{kappa}B-Luc, was transiently transfected into ARP-1 cells and the luciferase activity was measured. In a 48-hour treatment of ARP-1 cells with 10–7 mol/L dexamethasone, the relative luciferase activity was reduced by 70% compared with the nondexamethasone-treated control cells. Treatment of cells with 200 ng/mL of PCDGF/GP88 was able to prevent this dexamethasone-mediated down-modulation of luciferase activity.

Collectively, results from the above experiments indicate that the activation of MAPKs and PI3K pathways as well as the activation of NF-{kappa}B by PCDGF/GP88 all contribute to counter the apoptotic effect mediated by dexamethasone in ARP-1 cells.

Overexpression of PCDGF/GP88 in ARP-1 cells. To further investigate the effect of PCDGF/GP88 on dexamethasone sensitivity of ARP-1 cells, PCDGF/GP88 was overexpressed in ARP-1 cells. The empty vector (ARP-1-EV) and PCDGF/GP88–overexpressing ARP-1 cells (ARP-1-PCDGF/GP88) were selected in the presence of G418. A significantly higher level of the PCDGF/GP88 was expressed by ARP-1-PCDGF/GP88 cells compared with the control ARP-1-EV cells when examined by Western blot analysis (~6.8-fold above the control ARP-1-EV cells). When examined by a sandwich ELISA system, PCDGF/GP88 expression of ARP-1-PCDGF/GP88 cells (70 ng/106 cells) was ~9-fold higher than in the control ARP-1-EV cells (8 ng/106 cells).

Dexamethasone effect on ARP-1-PCDGF/GP88 cells. Because PCDGF/GP88 confers dexamethasone resistance in ARP-1 cells, experiments were carried out to examine whether dexamethasone (10–8-10–5 mol/L) could inhibit the proliferation of ARP-1-PCDGF/GP88 cells. Whereas dexamethasone inhibited the proliferation of ARP-1-EV cells in a dose-dependent fashion, ARP-1-PCDGF/GP88 cells were significantly resistant to dexamethasone treatment. Forty-eight-hour treatment with 10–5, 10–6, 10–7, and 10–8 mol/L dexamethasone inhibited the cell growth of ARP-1-EV cells by 65%, 58%, 52%, and 41%, respectively (Fig. 5A and C). However, for ARP-1-PCDGF/GP88 cells, the same treatment only inhibited the cell growth by 31%, 21%, 15%, and 11%, respectively (Fig. 5A and C). Similar result was observed in cell survival (Fig. 5B). When dexamethasone-induced (10–7 mol/L) PARP cleavage was examined, significantly less amount of PARP cleavage was observed in ARP-1-PCDGF/GP88 cells than in control ARP-1-EV cells after 48 hours of treatment (Fig. 5D). These data show that overexpression of PCDGF/GP88 in ARP-1 cells leads to dexamethasone resistance.



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  		Fig. 5. Effect of dexamethasone on ARP-1-PCDGF/GP88 and ARP-1-EV cell apoptosis and viability. A and B, dose response of effect of dexamethasone on AR-1-PCDGF/GP88 and ARP-1-EV cells: 4 x 104 cells were cultured in RPMI 1640 supplemented with 10% charcoal-treated FBS in the presence or absence of dexamethasone (10–5-10–8 mol/L) for 48 hours. Cell density (A) and viability (B) were examined after 48-hour treatment. C, time course effect of dexamethasone on ARP-1-PCDGF/GP88 and ARP-1-EV cells. Cell density was examined after 48-hour dexamethasone treatment. Columns, mean; bars, SD. D, Western blot analysis showing the effect of overexpression of PCDGF/GP88 on PARP cleavage as described in Materials and Methods. *, P < 0.05, compared with ARP-1-EV, con.

 
Glucocorticoid receptor function in ARP-1-PCDGF/GP88 and ARP-1-EV cells. Because ARP-1-PCDGF/GP88 cells are resistant to dexamethasone-mediated apoptosis, experiments were carried out to eliminate the possibility that this was due to the alterations in glucocorticoid receptor function in ARP-1-PCDGF/GP88 cells. For this purpose, we compared the ability of dexamethasone to stimulate GRE-luciferase reporter gene activity (16, 17) in ARP-1-EV and ARP-1-PCDGF/GP88 cells. Results from this experiment showed that 10–7 mol/L dexamethasone induced equivalent levels (5-fold stimulation) of GRE-luciferase activities in ARP-1-EV and ARP-1-PCDGF/GP88 cells. These results indicated that overexpression of PCDGF/GP88 did not affect glucocorticoid receptor function in ARP-1 cells and that the loss of dexamethasone responsiveness observed in ARP-1-PCDGF/GP88 cells was not due to a loss of glucocorticoid receptor function.

Colony-forming assay of ARP-1-EVand ARP-1-PCDGF/GP88 cells. The higher expression of PCDGF/GP88 is generally associated with increased tumorigenic properties of cells (10). A colony-forming assay was carried out to examine whether PCDGF/GP88–overexpressing ARP-1 cells had increased ability to form colonies in soft agar in the presence or absence of dexamethasone. As shown in Fig. 6A, the overexpression of PCDGF/GP88 in ARP-1 cells almost doubled the ability of cells to form colonies in soft agar compared with the control ARP-1-EV cells. Dexamethasone inhibited colony formation in ARP-1-EV cells by 70%, whereas it only inhibited ARP-1-PCDGF/GP88 cell colony formation by 16%. These data show that overexpression of PCDGF/GP88 promotes ARP-1 cells to form colonies in soft agar and renders them resistant to dexamethasone.



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  		Fig. 6. Colony formation and in vivo tumorigenesis assay of ARP-1-EV and ARP-1-PCDGF/GP88 cells. Cells (5,000) were plated in a soft agar medium in 35 mm dish in the presence or absence of 10–7 mol/L dexamethasone. Cells were incubated at 37°C with 5% CO2 for 3 weeks. The colonies were stained by 0.1% crystal violet and counted. ARP-1-EV and ARP-1-EV dexamethasone were considered as the control samples for ARP-1-PCDGF/GP88 and ARP-1-PCDGF/GP88 dexamethasone, respectively, in t test. Columns, mean; bars, SD. B, ARP-1-EV or ARP-1-PCDGF/GP88 cells were s.c. inoculated into athymic nude mice at 5 x 106 per site, four sites per mouse. There were five mice in each group. The tumor sizes were monitored everyday. The plot showed tumor size distribution in mice injected with ARP-1-EV and ARP-1-PCDGF/GP88 cells 22 days after mice were injected. *, P < 0.05, compared with corresponding control.

 
In vivo tumorigenicity study. The ability of PCDGF/GP88 to increase the tumorigenicity of ARP-1 cells was tested in vivo by comparing tumor formation of ARP-1-EV and ARP-1-PCDGF/GP88 injected into nude mice (Fig. 6B). Overexpression of PCDGF/GP88 dramatically increased the tumorigenicity when measured by tumor size, tumor weight, tumor incidence, and the day of appearance. The average tumor weight in ARP-1-PCDGF/GP88 cells inoculated mice was 1.6-fold higher compared with the tumors formed by ARP-1-EV cells. The tumor incidence in ARP-1-PCDGF/GP88 cells inoculated mice (16 tumors developed) was 5.3-fold higher than the control ARP-1-EV inoculated mice (three tumors developed). The visible appearance of tumor in ARP-1-PCDGF/GP88 inoculated mice was 3 days earlier than the control ARP-1-EV inoculated mice. These data indicate that the overexpression of PCDGF/GP88 increases tumorigenic property of ARP-1 cells.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Glucocorticoid analogues are used in the therapeutic regimen of multiple myeloma due to the fact that multiple myeloma cells are at least initially sensitive to glucocorticoid hormone. We have reported previously that PCDGF/GP88 is expressed in multiple myeloma cells and acts as an autocrine growth factor for these cells (11). In the present study, we investigated the role of PCDGF/GP88 in dexamethasone-induced apoptosis using dexamethasone-sensitive human multiple myeloma cell line ARP-1.

Glucocorticoid exerts pleiotropic proapoptotic effects in multiple myeloma cells, including caspase activation (detected by PARP cleavage assay), down-modulation of NF-{kappa}B, and inhibition of MAPK activation. PCDGF/GP88, on the other hand, exerts prosurvival and proliferative effects in multiple myeloma cells, including activations of PI3K-Akt and MAPK pathways. Therefore, it is logical to examine whether these two counteracting factors played a role in dexamethasone resistance. Although dexamethasone inhibited PCDGF/GP88 expression, results from our study indicated that PCDGF/GP88 could inhibit dexamethasone-induced cell death of multiple myeloma cells in a dose-dependent fashion. The observed PCDGF/GP88-mediated reduction in dexamethasone induced cell death could be due to either (a) the net effect of dexamethasone-induced cell death and PCDGF/GP88–induced enhanced proliferation and survival of multiple myeloma cells or (b) the effect of PCDGF/GP88 preventing dexamethasone-induced apoptosis. Flow cytometry analysis and PARP cleavage assay clearly showed that the PCDGF/GP88–mediated reversal of dexamethasone-induced cell death was through preventing dexamethasone-induced apoptosis. Our flow cytometry data showed that 200 ng/mL of PCDGF/GP88 significantly decreased the sub-G1 peak, detected as the hypodiploid population, induced by dexamethasone. When PARP cleavage, a marker of apoptosis, was examined, PCDGF/GP88 clearly showed the ability to prevent dexamethasone-induced PARP cleavage.

Known autocrine growth factors, such as interleukin-6 and insulin-like growth factor-I, have been shown to have the ability to prevent dexamethasone-induced apoptosis in multiple myeloma cells (13, 18). The antiapoptotic effect of insulin-like growth factor-I is mediated by the PI3K pathway, but not by the MAPK pathway (19). On the other hand, the antiapoptotic effect of interleukin-6 is not mediated via the PI3K pathway, as shown by the inability of PI3K inhibitors to prevent the antiapoptotic effect of interleukin-6 (19). We previously reported that PCDGF/GP88 activates MAPK and PI3K pathways in human multiple myeloma cells. The data from phosphorylation of MAPK/PI3K, PARP cleavage, and cell growth/survival showed that both signaling pathways contributed to the antiapoptotic effect of PCDGF/GP88 in multiple myeloma cells. Blockade of one signaling pathway leads to block most of the PCDGF/GP88 effect, which indicates that there is crosstalk between these two pathways. Our data show that NF-{kappa}B is involved in the antiapoptotic effect of PCDGF/GP88. This is consistent with other reports showing an association of dexamethasone-induced apoptosis with decreased NF-{kappa}B DNA binding and {kappa}B-dependent transcription (4). In multiple myeloma cells, our data indicate that the antiapoptotic effect of PCDGF/GP88 is mediated by MAPK, PI3K-Akt, and NF-{kappa}B pathways.

The overexpression of PCDGF/GP88 in dexamethasone-sensitive ARP-1 cells renders the cells insensitive to dexamethasone effect when compared with ARP-1-EV cells. The inhibitory effect of dexamethasone on cell proliferation viability was reduced in ARP-1-PCDGF/GP88 cells when compared with ARP-1EV cells. In addition, 10–7 mol/L dexamethasone only inhibited the colonies formed by ARP-1-PCDGF/GP88 cells by 16%, whereas the same concentration of dexamethasone inhibited the colonies formed by ARP-1-EV cell by 70%. PARP cleavage induced by dexamethasone was dramatically decreased in ARP-1-GP88 cells when treated by dexamethasone. This conclusion is compatible with our observations that PCDGF/GP88 can stimulate multiple myeloma cell growth/survival and that PCDGF/GP88 added exogenously can prevent dexamethasone-induced apoptosis. Consistent with this effect, we have shown previously that PCDGF/GP88 overexpression in breast cancer cells prevented tamoxifen-induced apoptosis (12). The PCDGF/GP88 effect on tumorigenesis of multiple myeloma cells was also examined using PCDGF/GP88–overexpressed ARP-1 cells. Overexpression of PCDGF/GP88 in multiple myeloma significantly increased colony formation in soft agar and dramatically increased tumorigenesis of the cells when injected into nude mice. Increased tumorigenesis of ARP-1 cells shown here was consistent with the reports that overexpression of PCDGF/GP88 increased the tumorigenesis of the human breast cancer cell line MCF-7 and of the human adrenal carcinoma cell line SW13 (20).

We have shown previously by immunohistochemical analysis of bone marrow smears obtained from multiple myeloma patients with anti-human PCDGF/GP88 antibody that PCDGF/GP88 was strongly expressed in multiple myeloma cells from patients having active disease whereas PCDGF/GP88 expression was negative in multiple myeloma patients in remission (11). In addition, we have shown previously that treatment of multiple myeloma cells with anti-PCDGF/GP88 antibody resulted in an inhibition of cell proliferation in vitro. Although these results do not provide direct information whether PCDGF/GP88 plays a role in vivo in multiple myeloma, it is a strong indication that PCDGF/GP88 level in multiple myeloma patients is associated with the presence of the disease and would suggest a possible role in vivo. Based on these reports and on the data presented here, it would be interesting to examine PCDGF/GP88 expression in bone marrow smears of patients before and after development of resistance to glucocorticoids.

PCDGF/GP88 expression in serum of healthy individuals was below detectable level using a PCDGF/GP88 ELISA kit developed in our laboratory with a detection limit at subnanogram per milliliter level.4 It was also reported that PCDGF/GP88 expression is higher in dexamethasone-insensitive multiple myeloma cell line RPMI 8226 than in dexamethasone-sensitive cell line ARP-1 (11). These data and the data presented here would suggest that PCDGF/GP88 may be a suitable biomarker for the development of novel therapy and diagnostic of dexamethasone-resistant human multiple myeloma.


   Acknowledgments
 
We thank Dr. Robert Fenton for the gift of multiple myeloma cells and Huifang Dai and Binbin Yue for preparation of PCDGF.


   Footnotes
 
Grant support: NIH grant 1RO1 CA 85367-01 and the Concern Foundation.

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.

Note: W. Wang is currently in Department of Medicine and Microbiology-Immunology, University of California, San Francisco, California.

4 Unpublished data. Back

Received 5/ 2/05; revised 10/ 9/05; accepted 10/20/05.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hallek M, Bergsagel PL, Anderson KC. Multiple myeloma: increasing evidence for a multistep transformation process. Blood 1998;91:3–21.[Free Full Text]
  2. Greenlee RT, Hill-Harmon MB, Murray T, Thun M. Cancer statistics, 2001. CA Cancer J Clin 2001;51:15–36.[Abstract/Free Full Text]
  3. Oken MM. Multiple myeloma: prognosis and standard treatment. Cancer Invest 1997;15:57–64.[Medline]
  4. Feinman R, Koury J, Thames M, Barlogie B, Epstein J, Siegel DS. Role of NF-{kappa}B in the rescue of multiple myeloma cells from glucocorticoid-induced apoptosis by bcl-2. Blood 1999;93:3044–52.[Abstract/Free Full Text]
  5. Smith ML, Newland AC. Treatment of myeloma. QJM 1999;92:11–4.[Free Full Text]
  6. Moalli PA, Rosen ST. Glucocorticoid receptors and resistance to glucocorticoids in hematologic malignancies. Leuk Lymphoma 1994;15:363–74.[Medline]
  7. Frassanito MA, Cusmai A, Iodice G, Dammacco F. Autocrine interleukin-6 production and highly malignant multiple myeloma: relation with resistance to drug-induced apoptosis. Blood 2001;97:483–9.[Abstract/Free Full Text]
  8. Zhou J, Gao G, Crabb JW, Serrero G. Purification of an autocrine growth factor homologous with mouse epithelin precursor from a highly tumorigenic cell line. J Biol Chem 1993;268:10863–9.[Abstract/Free Full Text]
  9. He Z, Ismail A, Kriazhev L, Sadvakassova G, Bateman A. Progranulin (PC-cell-derived growth factor/acrogranin) regulates invasion and cell survival. Cancer Res 2002;62:5590–6.[Abstract/Free Full Text]
  10. Serrero G. Autocrine growth factor revisited: PC-cell-derived growth factor (progranulin), a critical player in breast cancer tumorigenesis. Biochem Biophys Res Commun 2003;308:409–13.[CrossRef][Medline]
  11. Wang W, Hayashi J, Kim WE, Serrero G. PC cell-derived growth factor (granulin precursor) expression and action in human multiple myeloma. Clin Cancer Res 2003;9:2221–8.[Abstract/Free Full Text]
  12. Tangkeangsirisin W, Hayashi J, Serrero G. PC cell-derived growth factor mediates tamoxifen resistance and promotes tumor growth of human breast cancer cells. Cancer Res 2004;64:1737–43.[Abstract/Free Full Text]
  13. Hardin J, MacLeod S, Grigorieva I, et al. Interleukin-6 prevents dexamethasone-induced myeloma cell death. Blood 1994;84:3063–70.[Abstract/Free Full Text]
  14. Lu R, Serrero G. Stimulation of PC cell-derived growth factor (epithelin/granulin precursor) expression by estradiol in human breast cancer cells. Biochem Biophys Res Commun 1999;256:204–7.[CrossRef][Medline]
  15. Lu R, Serrero G. Mediation of estrogen mitogenic effect in human breast cancer MCF-7 cells by PC-cell-derived growth factor (PCDGF/granulin precursor). Proc Natl Acad Sci U S A 2001;98:142–7.[Abstract/Free Full Text]
  16. Krishnan AV, Swami S, Feldman D. Estradiol inhibits glucocorticoid receptor expression and induces glucocorticoid resistance in MCF-7 human breast cancer cells. J Steroid Biochem Mol Biol 2001;77:29–37.[CrossRef][Medline]
  17. Lee YN, Lee HY, Lee YM, et al. Involvement of glucocorticoid receptor in the induction of differentiation by ginsenosides in F9 teratocarcinoma cells. J Steroid Biochem Mol Biol 1998;67:105–11.[Medline]
  18. Xu F, Gardner A, Tu Y, Michl P, Prager D, Lichtenstein A. Multiple myeloma cells are protected against dexamethasone-induced apoptosis by insulin-like growth factors. Br J Haematol 1997;97:429–40.[CrossRef][Medline]
  19. Tu Y, Gardner A, Lichtenstein A. The phosphatidylinositol 3-kinase/AKT kinase pathway in multiple myeloma plasma cells: roles in cytokine-dependent survival and proliferative responses. Cancer Res 2000;60:6763–70.[Abstract/Free Full Text]
  20. He Z, Bateman A. Progranulin gene expression regulates epithelial cell growth and promotes tumor growth in vivo. Cancer Res 1999;59:3222–9.[Abstract/Free Full Text]




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