Cancer Research Cancer Epigenetics Protein Translation and Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Conze, D.
Right arrow Articles by Rincón, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Conze, D.
Right arrow Articles by Rincón, M.
[Cancer Research 61, 8851-8858, December 15, 2001]
© 2001 American Association for Cancer Research

Regular Articles

Autocrine Production of Interleukin 6 Causes Multidrug Resistance in Breast Cancer Cells1

Dietrich Conze, Linda Weiss, Patricia Susan Regen, Alok Bhushan, Donald Weaver, Peter Johnson and Mercedes Rincón2

Section of Immunobiology, Department of Medicine [D. C., L. W., P. S. R., M. R.] and Pathology Department [D. W.], University of Vermont, Burlington, Vermont 05405; Department of Pharmaceutical Sciences, College of Pharmacy, Idaho State University, Pocatello, Idaho 83209 [A. B.]; and Eukaryotic Transcriptional Regulation Section, Regulation of Cell Growth Laboratory, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702 [P. J.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It has been shown that serum levels of interleukin (IL)-6 are elevated in patients with various types of cancer. However, the exact source of IL-6 in these patients and its role in tumor progression remain unclear. Here we demonstrate that the autocrine production of IL-6 by tumor cells promotes resistance of the cells to chemotherapy, a novel function of IL-6 in cancer biology. Breast cancer cells that are sensitive to drug treatment do not express IL-6, whereas high levels of IL-6 are produced by multidrug-resistant breast cancer cells. Expression of the IL-6 gene in drug-sensitive breast cancer cells increases their resistance to drug treatment by activating the CCAAT enhancer-binding protein family of transcription factors and inducing mdr1 gene expression. Thus, the autocrine production of IL-6 by tumor cells is an important factor in determining the susceptibility or resistance of these cells to drug treatment. Because tumors from some breast cancer patients contain IL-6-producing cells, it is possible that IL-6 could potentially be used as a prognostic factor for chemotherapy resistance.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
IL-63 is a cytokine produced by a number of cell types including fibroblasts, macrophages, T and B lymphocytes, endothelial cells, glial cells, and keratinocytes in response to a variety of external stimuli (e.g., IL-1, tumor necrosis factor, and platelet-derived growth factor). IL-6 induces synthesis of acute phase response proteins in hepatocytes, terminal differentiation of B cells to antibody-producing plasma cells, differentiation of monocytes to macrophages, and growth of hematopoietic stem cells (for review, see Ref. 1 ). We have shown that IL-6 differentiates CD4+ T cells to effector T helper 2 cells (2 , 3) .

IL-6 binds to the cell surface IL-6R (IL-6R{alpha}), causing its dimerization with signal-transducing element gp130/IL-6Rß (4, 5, 6) . IL-6 can also bind to a soluble IL-6R (sIL-6R{alpha}) to induce IL-6 signal transduction pathways (7) . The gp130 element of the IL-6R is shared by the ciliary neutrophilic factor, oncostatin M, leukemia inhibitory factor, IL-11, and cardiotropin-1 receptors. Dimerization of the gp130 element in response to IL-6 leads to the activation of the MAPK signaling pathways, specifically, the extracellular signal-regulated kinases 1 and 2 (8) . IL-6 signaling also leads to the activation of the transcription factor C/EBPß [also defined as NFIL-6/IL-6DBP (9 , 10) ], which appears to be phosphorylated by MAPK (11) . The expression of C/EBP{delta} (NFIL-6ß), another member of the C/EBP family of transcription factors, is induced in response to IL-6 (12) . C/EBP{delta} can act synergistically with C/EBPß to increase the transcriptional activity of type 1 IL-6-responsive elements (13) .

gp130 dimerization induces the phosphorylation of associated JAK family members JAK1 and JAK2 and Tyk2 (14, 15, 16) . IL-6 has been shown to induce the tyrosine phosphorylation and nuclear translocation of STAT3 and, to a much lesser extent, STAT1. These transcription factors in turn bind to DNA and regulate the transcriptional activity of type 2 IL-6-responsive elements (17, 18, 19) . It has been recently shown that the induction of C/EBP{delta} by IL-6 is regulated by STAT3 (20) , suggesting that cross-talk exists between these two IL-6-mediated signaling pathways.

IL-6 has been involved in the growth of a variety of cancer cells (for review, see Ref. 1 ). It can act as a growth factor for myeloma/plasmacytoma, renal cell carcinoma, cervical carcinoma, AIDS Kaposi’s sarcoma-derived cells, and certain T- and B-cell lymphomas (21, 22, 23, 24, 25, 26) . Analysis of transgenic mice overexpressing IL-6 in B cells has shown that IL-6 is involved in the development of plasmacytoma and myeloma (1) . Moreover, elevated IL-6 serum levels have been detected in patients with renal cancer, ovarian cancer, plasmacytoma, and myeloma cancer (27) .

In the present study, we demonstrate that IL-6 can be produced by breast cancer cells in an autocrine fashion that confers their resistance to drug treatment but does not affect their growth. The IL-6-induced drug resistance is associated with increased expression of the multidrug resistance gene, mdr1, and up-regulation of C/EBPß and C/EBP{delta}. Because analysis of IL-6 expression shows that IL-6 can be produced by some tumor cells in breast cancer patients, it is possible that these IL-6-producing cancer cells are more resistant to chemotherapeutic agents.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines.
MCF7 and MCF7/ADR cells were a gift of Dr. Ken Cowan (National Cancer Institute, Bethesda, MD). MCF7, MCF7/ADR, MCF7/IL-6A, and MCF7/IL-6B cells were maintained in RPMI 1640 (Life Technologies, Inc., Gaithersburg, MD) containing 10% FCS (Life Technologies, Inc.). MCF7/ADR cells were maintained in the presence of 1 µM doxorubicin (Sigma Chemical Co., St. Louis, MO) to maintain the multidrug-resistant phenotype. Seven days before experiments were performed, MCF7/ADR cells were removed from doxorubicin-containing medium and maintained in doxorubicin-free medium as described previously (28 , 29) .

Recombinant human IL-6 (R&D Systems, Minneapolis, MN) was used to pretreat MCF7 cells. The cells were cultured in the presence of exogenous IL-6 (50 ng/ml) for 10 days. IL-6 was added to the culture every 2 days. After the pretreatment period, the cells were harvested, washed, and replated in the presence of IL-6, and their resistance to doxorubicin was determined by the MTT assay.

The human IL-6 cDNA plasmid (pBSF2.38.1) was kindly provided by Dr. Toshio Hirano (Biomedical Research Group, Osaka University Medical School, Osaka, Japan). The 1100-bp EcoRI-BamHI fragment obtained from pBSF2.38.1 was subcloned into the eukaryotic expression vector pcDNA3 (Invitrogen, Carlsbad, CA). MCF7 cells were transfected using Lipofectin reagent (Life Technologies, Inc.) following the manufacturer’s protocol. Transfected cells were then plated in RPMI 1640 containing 10% FCS and 400 µg/ml G418 (Life Technologies, Inc.) for 4 weeks, and stable transfectants were selected for G418 resistance. Selected clones were maintained in medium containing 400 µg/ml G418 and transferred to G418-free medium 1 week before the experiments.

MCF7, MCF7/ADR, MCF7/IL-6A, and MCF7/IL-6B cells were plated at equal densities (3.0 x 103 cells/well), and their proliferation rates were determined after 3 days by measurement of [3H]thymidine (Amersham Pharmacia Biotech Corp., Piscataway, NJ) incorporation.

Selection of Cells Expressing a Dominant Negative Mutant of C/EBP.
MCF7/IL-6A cells were cotransfected with the pHOOK-2 plasmid (Invitrogen) and either the empty vector (pCMV) or the expression vector containing the dominant negative C/EBP mutant (pCMV-A-C/EBP) using Lipofectin reagent following the manufacturer’s protocol. The transfection media were replaced with RPMI 1640 containing 10% FCS, and the cells were left in culture for 48 h. The Capture-Tec pHOOK-2 system (Invitrogen) was then used to select for the transfected cells following the manufacturer’s protocol.

Cytokine Measurements.
Cells were plated at equal densities (3.0 x 103 cells/well) in medium alone for 6 days. Supernatants were harvested at different time points, and IL-6 production was determined by ELISA. ELISAs were performed using a purified antihuman IL-6 MAb (2 µg/ml), the corresponding biotinylated antihuman IL-6 MAb (1 µg/ml; PharMingen, San Diego, CA), and horseradish peroxidase-conjugated avidin D (2.5 µg/ml; Vector Laboratories, Burlingame, CA) following the protocol recommended by the manufacturer (PharMingen). Recombinant human IL-6 (R&D Systems) was used as the standard.

RNA Isolation and Northern Blot Analysis.
Total RNA was extracted using the Ultraspec RNA isolation system (Biotecx Laboratories, Inc., Houston, TX). Total RNA (10 µg) was analyzed by Northern blot as described previously (30) . Northern blots were hybridized with cDNA probes specific for human IL-6 and human HPRT labeled with [{alpha}-32P]dCTP (Amersham Pharmacia Biotech) using the Random Primer Kit (Stratagene, La Jolla, CA).

RT-PCR.
Total RNA was extracted using the Ultraspec RNA isolation system (Biotecx). The first-strand cDNA was obtained by reverse transcription as described previously (31) . The reverse transcription mixture (1 µl) was assayed to detect human IL-6, HPRT, and mdr1 mRNA by RT-PCR. The primers used for the amplification of IL-6 have been described previously (32) . The primers used for the amplification of HPRT were 5' hHPRT (5'-GTTGGATATAAGCCAGACTTTGTTG-3') and 3' hHPRT (5'-AGCCTATAGACTATCAGTTCCCT-3'). The primers used for the amplification of mdr1 have been described previously (33) . For IL-6 mRNA quantitation by real-time PCR, total RNA was extracted using the Ultraspec RNA isolation system (Biotecx) and reverse-transcribed, and the cDNA was quantified using the TaqMan system (Applied Biosystems, Foster City, CA). Values for IL-6 mRNA were normalized to the relative quantity of 18S RNA in each sample. The primer and probe sets used for the amplification of the IL-6 mRNA and the 18S RNA control were supplied by Applied Biosystems, kit numbers 4309894P and 4310893E, respectively).

MTT Assay.
The dose response of the cell lines to doxorubicin was determined using the MTT assay as described previously (34) . The cells were incubated in the presence of different concentrations of doxorubicin, vincristine, or Taxol (0–10 µM) for 4 days. On day 4, the remaining cells were stained with MTT (Sigma Chemical Co.), the cell supernatants were aspirated, and 100 µl of DMSO were added to each well. The plates were shaken for 5 min, and the absorbances were quantified at 540 nm using a spectrophotometer (Biotek, Colchester, VT). Absorbances were normalized, and LD50 values were calculated by nonlinear regression. Normalized data and nonlinear regression curves were plotted graphically as a percentage of viable cells.

Nuclear Extracts and EMSAs.
"Mini" nuclear extracts were made from 6 x 105 cells as described previously (35 , 36) . Binding reactions for the EMSAs were performed using 2 µg of nuclear protein and a [{alpha}-32P]dCTP-end-labeled double-strand oligonucleotide as described previously (31) . The C/EBP oligonucleotide used in the reaction mixture was C/EBP sense (5'-GGACGTCACATTGCACAATCTTAATA-3'; Ref. 32 ). One µl/reaction of a nonspecific antiserum against glutathione S-transferase, a specific anti-C/EBPß peptide rabbit antiserum (37) , or an anti-C/EBP{delta} rabbit antiserum was used in supershift experiments. The anti-C/EBP{delta} polyclonal antiserum was raised against the NH2-terminal portion (amino acids 1–188) of murine C/EBP{delta}.4

Cell Surface Staining.
To determine the surface expression of gp130, MCF7 and MCF7/ADR cells were harvested, washed, and stained with a PE-conjugated antihuman gp130 MAb (PharMingen) or a PE-conjugated antihuman IgG1 MAb (PharMingen) as an isotype control and analyzed by flow cytometry.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Production of IL-6 by Multidrug-resistant Breast Cancer Cells.
Elevated serum levels of IL-6 have been described in some breast cancer patients (38) . In correlation with previous observations (39) , we have found that some tumor cells within human primary breast cancer tissue produce IL-6.5 Here we examined the production of IL-6 by the breast cancer cell line MCF7 and the multidrug-resistant breast cancer cell line MCF7/ADR. MCF7 and MCF7/ADR cells were plated at equal densities, cell supernatants were harvested at different time points, and the production of IL-6 by these cancer cells was determined by ELISA. No IL-6 was detected in the supernatant from MCF7 cells (Fig. 1A)Citation . In contrast, high levels of IL-6 were detected in the supernatant from MCF7/ADR cells (Fig. 1A)Citation .



View larger version (21K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. IL-6 production by MCF7 and MCF7/ADR cell lines. A, MCF7 and MCF7/ADR cells were plated in medium alone, and IL-6 production was determined by ELISA. B, total RNA was extracted from MCF7 and MCF7/ADR cells and examined for the expression of IL-6 and HPRT by Northern blot analysis. C, expression of IL-6 and HPRT was examined by RT-PCR using total RNA extracted from MCF7 and MCF7/ADR cells and primers specific for human IL-6 and HPRT. D, IL-6 mRNA levels in MCF7 and MCF7/ADR cells were determined using real-time PCR.

 
To determine whether the accumulation of IL-6 in the supernatant of the MCF7/ADR cells was due to the selective expression of IL-6 in these cells, we examined IL-6 gene expression by Northern blot analysis. High levels of IL-6 mRNA were present in MCF7/ADR cells, but no IL-6 mRNA was detected in MCF7 cells (Fig. 1B)Citation .

To confirm that the IL-6 gene is not expressed in MCF7 cells, we examined IL-6 gene expression in MCF7 and MCF7/ADR cells by RT-PCR. RT-PCR for HPRT was also performed as a positive control. In correlation with the results we obtained by Northern blot analysis, no IL-6 mRNA was detected in MCF7 cells despite expression of the HPRT gene (Fig. 1C)Citation . In contrast, high levels of IL-6 mRNA were detected MCF7/ADR cells, whereas the levels of HPRT mRNA were similar to those in MCF7 cells (Fig. 1C)Citation . Real-time PCR was also performed to quantify the levels of IL-6 gene expression in the MCF7/ADR cells. No IL-6 mRNA was detected in MCF7 cells, whereas high levels of IL-6 mRNA were present in MCF7/ADR cells (Fig. 1D)Citation . MCF7 cells fail to express the IL-6 gene, but expression of the IL-6 gene is significantly up-regulated in the drug-resistant MCF7/ADR cells.

Activation of C/EBPß and C/EBP{delta} in Multidrug-resistant Breast Cancer Cells.
The selective expression and secretion of IL-6 by MCF7/ADR cells revealed a potential association between the production of IL-6 and multidrug resistance. It was possible that the production of IL-6 could confer the resistance of MCF7/ADR cells to cytotoxic drugs by inducing IL-6 signal transduction pathways in an autocrine manner. We first examined the expression of the signal-transducing element of the IL-6R, gp130. MCF7 and MCF7/ADR cells were stained with a PE-conjugated anti-gp130 MAb and analyzed by flow cytometry. The histogram profiles depict the cell surface expression of the gp130 element on both MCF7 and MCF7/ADR cells (Fig. 2A)Citation . Comparable levels of gp130 were present on both MCF7 and MCF7/ADR cells (Fig. 2A)Citation , indicating that IL-6 was capable of mediating signals in both types of cells.



View larger version (30K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Activation of the IL-6 signal transduction pathway in MCF7/ADR cells. A, MCF7 and MCF7/ADR cells were harvested and stained with a PE-conjugated antihuman gp130 (IL-6Rß) MAb and examined by flow cytometry. Histograms represent the cell surface expression of gp130 (thick line). A PE-conjugated isotype control MAb (thin line) was used as a negative control. B, C/EBP DNA binding was examined by EMSA using nuclear extracts from MCF7 cells (M) and MCF7/ADR cells (A) using a 32P-end-labeled double-stranded oligonucleotide containing the C/EBP consensus binding site. The binding reactions were performed in the presence of nonspecific antiserum against glutathione S-transferase (control), a specific antiserum against C/EBPß, or an antiserum against C/EBP{delta} for supershift analysis. The arrow indicates the supershifted complex of C/EBPß or C/EBP{delta}.

 
IL-6 activates the transcription factors C/EBPß (NFIL-6) and C/EBP{delta} (NFIL-6ß; Refs. 9 , 10 , 12 , and 13 ). To determine whether the IL-6 pathway was activated in MCF7/ADR cells, the DNA binding of C/EBP transcription factors was determined by EMSA. Nuclear extracts from MCF7 and MCF7/ADR cells were incubated with an oligonucleotide containing the consensus regulatory sequence recognized by the C/EBP family of transcription factors (32) . Elevated levels of C/EBP DNA complexes were observed in MCF7/ADR cells as compared with MCF7 cells (Fig. 2B)Citation . To identify the components of the C/EBP complexes, we performed EMSA experiments in the presence of antiserum against the C/EBPß and C/EBP{delta} transcription factors. Supershift analysis using anti-C/EBP{delta} antiserum showed high levels of C/EBP{delta} in the nucleus of MCF7/ADR cells, whereas no C/EBP{delta} was detected in MCF7 cells. Elevated levels of C/EBPß were also present in the nucleus of MCF7/ADR cells as compared with MCF7 cells (Fig. 2B)Citation . These results demonstrate that this IL-6 signal transduction pathway is constitutively activated in MCF7/ADR cells characterized by the selective accumulation of C/EBPß and C/EBP{delta} in the nucleus. Furthermore, these results suggest that MCF7/ADR cells undergo persistent IL-6-mediated signaling in an autocrine manner.

IL-6 Confers Multidrug Resistance in MCF7 Cells.
Multidrug resistance is the broad spectrum resistance of human cancers to potent anticancer agents (40 , 41) . To date, the molecular mechanisms by which tumor cells become resistant to diverse groups of drugs remain unclear. The selective production of IL-6 by the MCF7/ADR cells suggested that IL-6 could play a role in the resistance of these cells to the cytotoxic activities of anticancer compounds. To address this hypothesis, MCF7 cells were cultured in the presence or absence of IL-6 for 10 days. After the pretreatment period, the cells were harvested, washed, and replated in the presence or absence of IL-6, and their resistance to doxorubicin was determined by the MTT assay (34) . Pretreatment of MCF7 cells with IL-6 caused an 8–10 fold increase in resistance to doxorubicin (Fig. 3)Citation , indicating that the presence of exogenous IL-6 increased the resistance of breast cancer cells to doxorubicin treatment.



View larger version (19K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Treatment with IL-6 increased the resistance of MCF7 cells. MCF7 cells were cultured in the presence (MCF7/preIL-6) or absence (MCF7) of exogenous IL-6 for 10 days. After the pretreatment period, the cells were replated in the presence or absence of IL-6, and their resistance to doxorubicin was determined by the MTT assay.

 
To determine whether the endogenous production of IL-6 by tumor cells could provide self-protection against drug-induced cell death, we constitutively expressed IL-6 in MCF7 cells and examined the effect of IL-6 expression on the drug resistance of these cells. MCF7 cells were transfected with an expression vector containing the human IL-6 gene. Stable MCF7 transfected (MCF7/IL-6) clones were isolated and screened for their ability to produce IL-6. Two representative clones that produced intermediate (MCF7/IL-6A) and high (MCF7/IL-6B) levels of IL-6 (Fig. 4A)Citation were chosen for subsequent studies. The levels of IL-6 gene expression in the two stable transfected clones were also quantified by real-time PCR. In correlation with the secreted IL-6 levels, high levels of IL-6 mRNA were detected in the MCF7/IL-6B clone, and low levels of IL-6 mRNA were detected in the MCF7/IL-6A clone (Fig. 4B)Citation .



View larger version (30K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. Expression of IL-6 in MCF7 cells increases resistance to doxorubicin. A, MCF7, MCF7/ADR, and MCF7 cells from two IL-6-transfected MCF7 clones (MCF7/IL-6A and MCF7/IL-6B) were plated, and IL-6 production was determined by ELISA. B, IL-6 mRNA levels in MCF7/IL-6A and MCF7/IL-6B cells were quantified using real-time PCR. C, MCF7 (LD50 = 0.0567 µM), MCF7/ADR (LD50 = 16.4 µM), MCF7/IL-6A (LD50 = 4.04 µM), and MCF7/IL-6B (LD50 = 4.07 µM) cells were plated out, and the resistance of the cells to doxorubicin was determined by the MTT assay. D and E, MCF7, MCF7/ADR, and MCF7/IL-6A cells were plated out, and the resistance of the cells to vincristine (D) or Taxol (E) was determined by the MTT assay. F, MCF7, MCF7/ADR, MCF7/IL-6A, and MCF7/IL-6B cells were plated, and their rate of proliferation was determined by [3H]thymidine incorporation.

 
To determine whether the expression of IL-6 in MCF7 cells could increase their sensitivity to drug treatment, we examined the susceptibility or resistance of MCF7/IL-6A and MCF7/IL-6B cells to doxorubicin treatment using the MTT assay. Interestingly, MCF7/IL-6A and MCF7/IL-6B cells exhibited an approximately 70-fold increase in their resistance to doxorubicin as compared with MCF7 cells (Fig. 4C)Citation . Although the levels of IL-6 secreted by MCF7/IL-6A were lower than those secreted by MCF7/IL-6B, both cell lines were equally resistant to doxorubicin treatment, suggesting that low levels of IL-6 produced by tumor cells are sufficient to confer drug resistance. Stable MCF7 transfected clones that did not produce IL-6 exhibited similar drug sensitivity to MCF7 cells (data not shown).

To determine whether the endogenous production of IL-6 can confer resistance to other drugs associated with multidrug resistance, we examined the susceptibility or resistance of MCF7, MCF7/ADR, and MCF7/IL-6A cells to vincristine and Taxol (42) . Similar to the results with doxorubicin, MCF7/ADR and MCF7/IL-6A cells exhibited increased resistance to both vincristine (Fig. 4D)Citation and Taxol (Fig. 4E)Citation as compared with MCF7 cells. In contrast, MCF7, MCF7/IL-6A, and MCF7/ADR cells were equally sensitive to 5-fluorouracil, a drug not associated with multidrug resistance (data not shown). Thus, the production of IL-6 by breast cancer cells promotes multidrug resistance to cytotoxic drugs.

Because IL-6 has been involved in cell growth, we addressed whether this effect was due to increased cell growth. We examined the proliferation of MCF7, MCF7/ADR, MCF7/IL-6A, and MCF7/IL-6B cells by [3H]thymidine incorporation. The proliferation observed in MCF7/IL-6A and MCF7/IL-6B cells was similar to that observed in MCF7 cells (Fig. 4F)Citation . These data therefore demonstrate that the autocrine production of IL-6 by breast cancer cells does not promote cell growth but enhances cell resistance to drug-induced cell death.

Endogenous Production of IL-6 in Breast Cancer Cells Induces mdr1 Gene Expression by Activating C/EBP Transcription Factors.
The selective expression of a Mr 170,000 transmembrane glycoprotein, P-gp, has been associated with the multidrug resistance phenotype of cancer cells. P-gp is an energy-dependent transmembrane pump encoded for by the mdr1 gene and is believed to reduce the intracellular accumulation of anticancer drugs (43, 44, 45, 46, 47, 48, 49, 50, 51, 52) . It has been previously demonstrated that MCF7/ADR cells, but not MCF7 cells, express high levels of mdr1 and its gene product, P-gp (45) . To test whether the enhanced drug resistance of the MCF7/IL-6 cells was also associated with the presence of P-gp, we examined mdr1 expression in MCF7, MCF7/ADR, MCF7/IL-6A, and MCF7/IL-6B cells by RT-PCR. In correlation with previous studies (45) , no mdr1 gene expression was detected in the MCF7 cells, but high levels were expressed in MCF7/ADR cells (Fig. 5A)Citation . Interestingly, high levels of mdr1 mRNA were found in MCF7/IL-6A and MCF7/IL-6B cells (Fig. 5A)Citation . These data indicate that the autocrine production of IL-6 induces mdr1 gene expression in breast cancer cells. P-gp protein levels were also elevated in MCF7/IL-6A and MCF7/IL-6B cells (data not shown).



View larger version (60K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. Induction of C/EBPß and C/EBP{delta} and mdr1 gene expression in MCF7/IL-6 cells causes mdr1 expression. A, expression of mdr1 and HPRT was examined by RT-PCR using total RNA extracted from MCF7, MCF7/ADR, MCF7/IL-6A, and MCF7/IL-6B cells and primers specific for human mdr1 and HPRT as described in "Materials and Methods." B, C/EBP DNA binding was examined by EMSA using nuclear extracts from MCF7 (M), MCF7/ADR (A), MCF7/IL-6 (M/A), and MCF7/IL-6B (M/B) cells and a 32P-end-labeled double-stranded oligonucleotide containing the C/EBP consensus binding site. The binding reactions were performed in the presence of nonspecific antiserum against glutathione S-transferase (Control), a specific antiserum against C/EBPß, or an antiserum against C/EBP{delta} for supershift analysis. The arrow indicates the supershifted complex of C/EBPß or C/EBP{delta}. C, mdr1 and HPRT gene expression was examined by RT-PCR using total RNA extracted from MCF7/IL-6A cells transiently transfected with either the empty vector (-) or the vector containing a dominant negative mutant of C/EBP (A-C/EBP).

 
Previous reports have shown that a C/EBP consensus binding site is present in the mdr1 promoter region and is able to mediate mdr1 gene transcription (53 , 54) . In correlation, our results showed increased levels of C/EBP complexes present in the MCF7/ADR (Fig. 2B)Citation cells that express mdr1. To determine whether the increase in mdr1 expression in MCF7/IL-6A and MCF7/IL-6B cells was due to the activation of C/EBP transcription factors by IL-6, we examined the DNA binding of C/EBP transcription factors by EMSA. Similar to MCF7/ADR cells, high levels of C/EBP complexes were present in the nucleus of MCF7/IL-6A and MCF7/IL-6B cells as compared with MCF7 cells (Fig. 5B)Citation . Supershift analysis using anti-C/EBP{delta} and anti-C/EBPß antisera showed high levels of nuclear C/EBP{delta} in MCF7/IL-6A and MCF7/IL-6B cells, whereas no C/EBP{delta} was detected in MCF7 cells. Moreover, the levels of nuclear C/EBPß in MCF7/IL-6A and MCF7/IL-6B cells were substantially higher than those present in MCF7 cells (Fig. 5B)Citation . These results suggest that the autocrine production of IL-6 by breast cancer cells activates C/EBP transcription factors, induces the expression of the mdr1 gene, and increases cell resistance to drug treatment.

To determine whether a causal relationship exists between the high amounts of C/EBP in the nuclei of the IL-6-producing MCF7 clones and the induction of mdr1 gene expression, we examined the effect of C/EBP inhibition on mdr1 gene expression in the MCF7/IL-6A cells. We performed transient transfections with a mutant form of C/EBP (A-C/EBP) that acts as a dominant negative for endogenous C/EBP (55 , 56) . MCF7/IL-6A cells transfected with either the A-C/EBP or the empty vector were isolated with the pHOOK-2 system. RNA was extracted, and mdr1 gene expression was determined by RT-PCR. Compared with the levels of mdr1 in cells transfected with the empty vector, expression of the A-C/EBP mutant in the MCF7/IL-6A cells caused a reduction of mdr1 gene expression (Fig. 5C)Citation . The partial inhibition of mdr1 gene expression by A-C/EBP correlates with the partial inhibitory effect of this mutant on the endogenous C/EBP DNA binding (data not shown). Thus, IL-6 up-regulates mdr1 gene by activation of the C/EBP signaling pathway.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
IL-6 is a pleiotropic cytokine that plays a significant role in the growth and differentiation of cells. Several studies have addressed the role of IL-6 in tumor cell growth, but its exact role remains varied and unclear. It appears that the effect of IL-6 on tumor cell growth may depend on the tumor cell type. In this study, we demonstrate for the first time that IL-6 plays a new role in cancer biology; it promotes multidrug resistance.

Chemotherapy has been one of the most effective and widely used means of treating cancer. The development of multidrug resistance, however, has posed major obstacles to the efficacy of chemotherapy and hence cancer treatment (57) . Here, we show that IL-6 is expressed and secreted by multidrug-resistant breast cancer cells, whereas no IL-6 gene expression is found in the parental drug-sensitive cells. Despite the expression of a large number of genes selectively expressed in multidrug-resistant cells, only a few of these genes have been shown to increase resistance to chemotherapeutic drugs (for review, see Ref. 58 ). We demonstrate that the expression of IL-6 in non-IL-6-producing breast tumor cells induces drug resistance, indicating that the production of IL-6 protects the cells from cytotoxic agents. Other studies have shown that inhibition of IL-6 secretion in prostate cancer cell lines increases the sensitivity of these cells to anticancer drugs (59) . Thus, some tumor cells may acquire the ability to express and produce IL-6 as a protective mechanism against drug-induced death. The stimuli responsible for the constitutive expression of IL-6 in multidrug-resistant cells remain to be determined.

In addition to the effect of endogenous production of IL-6, we have also shown that a relatively short period of treatment with exogenous IL-6 increases resistance of the MCF7 drug-sensitive cells to drug treatment but does not affect cell growth. These results suggest that administration of IL-6 to breast cancer patients could increase the survival of tumor cells during chemotherapy. Despite this prediction, IL-6 has been administered (Phase I/II clinical trials) to breast, ovarian, myeloid leukemic, and metastatic cancer patients in combination with chemotherapy. The principal rationale for administering IL-6 during these studies was to stimulate platelet growth and differentiation (60 , 61) , although no significant effects on platelet numbers were observed (61 , 62) . Other clinical studies have also used IL-6 as an antitumor agent in the treatment of renal cell carcinoma and melanoma patients (63, 64, 65) . It is possible that treatment with IL-6 contributed to the development of multidrug resistance in those patients.

The first and most widely studied factor known to modulate multidrug resistance is P-gp. P-gp is encoded by the mdr1 gene and is believed to mediate multidrug resistance by reducing the intracellular accumulation of cytotoxic drugs and compounds. Many types of cancers express high levels of mdr1 and are known to be unresponsive to chemotherapy (66, 67, 68) . In fact, P-gp expression has been considered to be a predictive factor of poor response to chemotherapy and overall survival (69, 70, 71) . Here we demonstrate that expression of IL-6 in drug-sensitive breast cancer cells induces mdr1 gene expression and, in correlation, increases the resistance of the cells to doxorubicin treatment. Thus, the induction of mdr1 gene expression is a potential mechanism by which IL-6 provides drug protection.

Specific regulatory elements have been identified within the 4.7-kb promoter region of the mdr1 gene (for review, see Ref. 72 ; see also Refs. 73 and 74 ), but the relative contribution of these elements to mdr1 gene expression remains unclear. There is a C/EBPß/NFIL-6-specific regulatory element located between -157 and -126 bp in the mdr1 promoter (53) . It has been shown that in response to phorbol 12-myristate 13-acetate, C/EBPß, a downstream component of the IL-6 signal transduction pathway (10) , can bind and transactivate the human mdr1 gene promoter (53 , 54) . C/EBP{delta}, another member of the C/EBP family of transcription factors, has been shown to act synergistically with C/EBPß from C/EBP consensus sequences and is also regulated by the IL-6 signal transduction pathway (13) . Here we demonstrate that the induction of mdr1 gene expression by IL-6 in breast cancer cells correlates with the activation of C/EBPß and C/EBP{delta}. Thus, the constitutive expression of the mdr1 gene in multidrug-resistant cells can be maintained by activated C/EBPß and C/EBP{delta}. We propose that some tumor cells may acquire the ability to express and produce IL-6. This ability may in turn act as a protective mechanism against chemotherapeutic drugs by activating C/EBPß/C/EBP{delta} and the expression of the mdr1 gene.

STAT3 is an additional transcription factor in the IL-6 signal transduction pathway (for review, see Ref. 1 ). After the treatment of cells with IL-6, gp130 dimerizes, and the gp130-associated JAK1 and JAK2 phosphorylate STAT3 (14 , 15) . Recently, it has been demonstrated that constitutively activated STAT3 leads to the transformation of cells, suggesting that STAT3 is an oncogenic factor (75) . In our study, we show that the expression of IL-6 in non-IL-6-producing breast cancer cells does not affect cell growth but does increase drug resistance. It is therefore possible that activation of the JAK/STAT signaling pathway in cells treated with IL-6 leads to the transformation of cells, whereas activation of the MAPK/C/EBP signaling pathway by IL-6 increases resistance to the cytotoxic activities of specific agents. Interestingly, the induction of the MAPK signaling pathway by IL-6 requires ErbB2/neu, a growth factor receptor implicated in the neoplastic transformation of prostate carcinoma cells, whereas activation of the JAK/STAT pathway is independent of ErbB2 (76) . Increased expression of ErbB2 has also been correlated with the resistance of cells to cytotoxic drugs (77, 78, 79, 80) .

In correlation with other studies (39) , we have observed that some tumor cells within primary breast cancer tissue have acquired the ability to produce IL-6.5 Additional studies need to be performed to ascertain whether a correlation exists between the presence of IL-6-producing breast cancer cells and the response of patients to chemotherapy. IL-6 could therefore be used as a predictive factor for the development of multidrug resistance and poor response to chemotherapy. Consistent with this hypothesis, elevated IL-6 serum levels have been found in ovarian cancer patients who do not respond to chemotherapy (81) . Elevated levels of IL-6 in serum have also been correlated with prostate metastasis and morbidity (82, 83, 84) It is clear that IL-6 should not be provided to cancer patients as a therapeutic agent, and drugs used to inhibit the expression of IL-6 or the IL-6 signal transduction pathway may be beneficial.


   ACKNOWLEDGMENTS
 
We thank Dr. Toshio Hirano for providing human IL-6 cDNA (pBSF2.38.1), Dr. Charles Vinson (Laboratory of Metabolism, National Cancer Institute) for providing pCMV-A-C/EBP expression vector, Collete Charland for flow cytometry analysis and helpful discussion, and Tim Hunter for assistance with the real-time PCR analysis.


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

1 Supported by a Gustavus and Louise Pfeiffer Research Foundation grant. Back

2 To whom requests for reprints should be addressed, at Department of Medicine/Immunobiology Section, Given Medical Building D-305, University of Vermont, Burlington, VT 05405. Phone: (802) 656-0937; Fax: (802) 656-3854; E-mail: mrincon{at}zoo.uvm.edu Back

3 The abbreviations used are: IL, interleukin; C/EBP, CCAAT enhancer-binding protein; MAPK, mitogen-activated protein kinase; JAK, Janus protein kinase; STAT, signal transducer and activator of transcription; MTT, microculture tetrazolium; PE, phycoerythrin; MAb, monoclonal antibody; P-gp, P-glycoprotein; HPRT, hypoxanthine phosphoribosyltransferase; RT-PCR, reverse transcription-PCR; EMSA, electrophoretic mobility shift assay; IL-6R, IL-6 receptor. Back

4 C. A. Cantwell and P. F. Johnson, unpublished observations. Back

5 L. Weiss and M. Rincón, unpublished data. Back

Received 3/29/01. Accepted 10/18/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hirano T. Interleukin 6 and its receptor: ten years later. Int. Rev. Immunol., 16: 249-284, 1998.[Medline]
  2. Rincón M., Anguita J., Nakamura T., Fikrig E., Flavell R. A. IL-6 directs the differentiation of IL-4-producing CD4+ T cells. J. Exp. Med., 185: 461-469, 1997.[Abstract/Free Full Text]
  3. Diehl S., Anguita J., Hoffmeyer A., Zapton T., Ihle J. N., Fikrig E., Rincon M. Inhibition of Th1 differentiation by IL-6 is mediated by SOCS1. Immunity, 13: 805-815, 2000.[Medline]
  4. Taga T., Hibi M., Hirata Y., Yamasaki K., Yasukawa K., Matsuda T., Hirano T., Kishimoto T. Interleukin-6 triggers the association of its receptor with a possible signal transducer, gp 130. Cell, 58: 573-581, 1989.[Medline]
  5. Hibi M., Murakami M., Saito M., Hirano T., Taga T., Kishimoto T. Molecular cloning and expression of an IL-6 signal transducer, gp 130. Cell, 63: 1149-1157, 1990.[Medline]
  6. Yamasaki K., Taga T., Hirata Y., Yawata H., Kawanishi Y., See B., Tanaiguchi T., Hirano T., Kishimoto T. Cloning and expression of human interleukin-6 (BSF-2/IFNb2) receptor. Science (Wash. DC), 241: 825-828, 1988.[Abstract/Free Full Text]
  7. Scambia G., Testa U., Benedetti P., Martucci R., Foti E., Petrini M., Amoroso M., Masciullo V., Peschle C., Mancuso S. Interleukin-6 serum levels in patients with gynecological tumors. Int. J. Cancer, 57: 318-323, 1994.[Medline]
  8. Daeipour M., Kumar G., Amaral M., Nel A. Recombinant IL-6 activates p42 and p44 mitogen-activated kinases in the IL-6 responsive B cell line, AF-10. J. Immunol., 150: 4743-4753, 1993.[Abstract]
  9. Akira S., Issihiki H., Sugita T., Tanabe O., Kinoshita S., Nishio Y., Nakajima T., Hirano T., Kishimoto T. A nuclear factor for IL-6 expression (NF-IL6) is a member of a C/EBP family. EMBO J., 9: 1897-1906, 1990.[Medline]
  10. Poli V., Mancini F. P., Cortese R. IL-6DBP, a nuclear protein involved in interleukin-6 signal transduction, defines a new family of leucine zipper proteins related to C/EBP. Cell, 63: 643-653, 1990.[Medline]
  11. Nakajima T., Kinoshita S., Sasagawa T., Sasaki K., Naruto M., Kishimoto T., Akira S. Phosphorylation at threonine-235 by a ras-dependent mitogen-activated protein kinase cascade is essential for transcription factor NF-IL6. Proc. Natl. Acad. Sci. USA, 90: 2207-2211, 1993.[Abstract/Free Full Text]
  12. Ramji D. P., Vitelli A., Tronche F., Cortese R., Ciliberto G. The two C/EBP isoforms, IL-6DBP/NFIL-6 and C/EBP{delta}/NFIL-6ß, are induced by IL-6 to promote acute phase gene transcription via different mechanisms. Nucleic Acids Res., 21: 289-294, 1993.[Abstract/Free Full Text]
  13. Kinoshita S., Akira S., Kishimoto T. A member of the C/EBP family, NFIL-6ß, forms a heterodimer and transcriptionally synergizes with NFIL-6. Proc. Natl. Acad. Sci. USA, 89: 1473-1476, 1992.[Abstract/Free Full Text]
  14. Lutticken C., Wegenka U., Yuan J., Buschmann J., Schindler C., Ziemiecki A., Harpur A., Wilks A., Yasukawa K., Taga T., Kishimoto T., Barbieri G., Pellegrini S., Sendtner M., Hienrich P., Horn F. Association of transcription factor APRF and protein kinase Jak1 with the interleukin-6 signal transducer gp130. Science (Wash. DC), 263: 89-92, 1993.
  15. Stahl N., Boulton T. G., Farruggella T., Ip N. Y., Davis S., Witthuhn B. A., Quelle F. W., Silvennoinen O., Barbieri G., Pellegrini S., et al Association and activation of Jak-Tyk kinases by CNTF-LIF-OSM-IL-6 ß receptor components. Science (Wash. DC), 263: 92-95, 1994.[Abstract/Free Full Text]
  16. Matsuda T., Yamanaka Y., Hirano T. Interleukin-6-induced tyrosine phosphorylation of multiple proteins in murine hematopoietic lineage cells. Biochem. Biophys. Res. Commun., 200: 821-828, 1994.[Medline]
  17. Akira S., Nishio Y., Inoue M., Wang X., Wei S., Matsusaka T., Yoshida K., Sudo T., Naruto M., Kishimoto T. Molecular cloning of APRF, a novel IFN-stimulated gene factor 3 p91-related transcriptional factor involved in the gp130 stimulated signaling pathway. Cell, 77: 63-71, 1994.[Medline]
  18. Fujitani Y., Nakajima K., Kojima H., Nakae K., Takeda T., Hirano T. Transcriptional activation of the IL-6 responsive element in the junB promoter is mediated by multiple Stat family proteins. Biochem. Biophys. Res. Commun., 202: 1181-1187, 1994.[Medline]
  19. Nakajima K., Matsuda T., Fujitani Y., Kojima H., Yamanaka Y., Nakae K., Takeda T., Hirano T. Signal transduction through the IL-6 receptor: involvement of multiple protein kinases, Stat factors, and a novel H7-sensitive pathway. Ann. N. Y. Acad. Sci., 762: 55-70, 1995.[Medline]
  20. Cantwell C., Sterneck E., Johnson P. Interleukin-6-specific activation of the C/EBP{delta} gene in hepatocytes is mediated by Stat3 and Sp1. Mol. Cell. Biol., 18: 2108-2117, 1998.[Abstract/Free Full Text]
  21. Kawano M., Hirano T., Matsuda T., Taga T., Horii Y., Iwato K., Asaoku H., Tang B., Tanabe O., Tanaka H., et al Autocrine generation and requirement of BSF-2/IL-6 for human multiple myelomas. Nature (Lond.), 332: 83-85, 1988.[Medline]
  22. Miki S., Iwano M., Miki Y., Yamamoto M., Tang B., Yokokawa K., Sonoda T., Hirano T., Kishimoto T. Interleukin-6 (IL-6) functions as an in vitro autocrine growth factor in renal cell carcinomas. FEBS Lett., 250: 607-610, 1989.[Medline]
  23. Eustace D., Han X., Gooding R., Rowbottom A., Riches P., Heyderman E. Interleukin-6 (IL-6) functions as an autocrine growth factor in cervical carcinomas in vitro. Gynecol. Oncol., 50: 15-19, 1993.[Medline]
  24. Miles S. A., Rezai A. R., Salazar-Gonzalez J. F., Van der Meyden M., Stevens R. H., Logan D. M., Mitsuyasu R. T., Taga T., Hirano T., Kishimoto T., et al AIDS Kaposi sarcoma-derived cells produce and respond to interleukin 6. Proc. Natl. Acad. Sci. USA, 87: 4068-4072, 1990.[Abstract/Free Full Text]
  25. Shimizu S., Hirano T., Yoshioka R., Sugai S., Matsuda T., Taga T., Kishimoto T., Konda S. Interleukin-6 (B-cell stimulatory factor 2)-dependent growth of a Lennert’s lymphoma-derived T-cell line (KT-3). Blood., 72: 1826-1828, 1988.[Abstract/Free Full Text]
  26. Yee C., Biondi A., Wang X. H., Iscove N. N., de Sousa J., Aarden L. A., Wong G. G., Clark S. C., Messner H. A., Minden M. D. A possible autocrine role for interleukin-6 in two lymphoma cell lines. Blood, 74: 798-804, 1989.[Abstract/Free Full Text]
  27. Snick J. Interleukin-6: an overview. Annu. Rev. Immunol., 8: 253-278, 1990.[Medline]
  28. Bichat F., Solis-Recendez G., Poullian M-G., Poupon M-F., Khayat D., Bastian G. S9788 modulation of P-glycoprotein- and multidrug-related protein-mediated multidrug resistance by Serier 9788 in doxorubicin-resistant MCF7 cells. Biochem. Pharmacol., 56: 497-502, 1998.[Medline]
  29. Gupta K. P., Ward N. E., Gravitt K. R., Bergman P. J., O’Brian C. A. Partial reversal of multidrug resistance in human breast cancer cells by an N-myristolated protein kinase C-{alpha} pseudosubstrate peptide. J. Biol. Chem., 271: 2102-2111, 1996.[Abstract/Free Full Text]
  30. Rincón M., Tugores A., López-Rivas A., Silva A., Alonso M., de Lándazuri M. O., López-Botet M. Prostaglandin E2 and the increase of intracellular cAMP inhibit the expression of interleukin 2 receptors in human T cells. Eur. J. Immunol., 18: 1791-1796, 1988.[Medline]
  31. Rincón M., Dérijard B., Chow C-W., Davis R. J., Flavell R. A. Reprogramming the signaling requirement for AP-1 (activator protein-1) activation during differentiation of precursor CD4+ T cells to effector Th1 and Th2 cells. Genes Funct., 1: 51-68, 1997.[Medline]
  32. Kiehntopf M., Hermann F., Brach M. A. Functional NF-IL-6/CCAAT enhancer-binding protein is required for tumor necrosis factor {alpha}-inducible expression of the granulocyte colony-stimulating factor (CSF), but not the granulocyte/macrophage CSF or interleukin 6 gene in human fibroblasts. J. Exp. Med., 181: 793-798, 1995.[Abstract/Free Full Text]
  33. Noonan K., Beck C., Holzmayer T., Chin J., Wunder J., Andrulis I., Gazdar A., Willman C., Griffith B., Hoff D. V., et al Quantitative analysis of MDR1 (multidrug resistance) gene expression in human tumors by polymerase chain reaction. Proc. Natl. Acad. Sci. USA, 87: 7160-7164, 1990.[Abstract/Free Full Text]
  34. Alley M. C., Scudiero D. A., Monks A., Hursey M. L., Czerwiniski M. J., Fine D. L., Abbott B. J., Mayo J. G., Shoemaker R. H., Boyd M. R. Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res., 48: 589-601, 1988.[Abstract/Free Full Text]
  35. Schreiber E., Matthias P., Müller M. M., Shaffner W. Rapid detection of octamer binding proteins with "mini"-extracts prepared from a small number of cells. Nucleic Acids Res., 17: 6419 1989.[Free Full Text]
  36. Tugores A., Alonso M. A., Sánchez-Madrid F., de Landázuri M. O. Human T cell activation through the activation-inducer molecule/CD69 enhances the activity of transcription factor AP-1. J. Immunol., 148: 2300-2306, 1992.[Abstract]
  37. Williams S., Baer M., Dillner A., Johnson P. CRP2 (C/EBP ß) contains a bipartite regulatory domain that controls transcriptional activation, DNA binding and cell specificity. EMBO J., 14: 3170-3183, 1995.[Medline]
  38. Zhang G., Adachi I. Serum interleukin-6 levels correlate to tumor progression and prognosis in metastatic breast carcinoma. Anticancer Res., 189: 1427-1432, 1999.
  39. Basolo F., Fiore L., Fontanini G., Conaldi P., Calvo S., Falcone V., Toniolo A. Expression of and response to interleukin-6 (IL6) in human mammary tumors. Cancer Res., 56: 3118-3122, 1996.[Abstract/Free Full Text]
  40. Biedler J. L., Riehm H. Cellular resistance to actinomysin D in Chinese hamster cells in vitro: cross resistance, radioautographic and cytogenic studies. Cancer Res., 30: 1174-1184, 1970.[Abstract/Free Full Text]
  41. Riordan J. R., Ling V. Genetic and biochemical characterization of multidrug resistance. Pharmacol. Ther., 28: 51-75, 1985.[Medline]
  42. Germann U. A. P-glycoprotein: a mediator of multidrug resistance in tumour cells. Eur. J. Cancer, 32A: 927-944, 1996.
  43. Juliano R. L., Ling V. A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. Biochim. Biophys. Acta, 455: 152-162, 1976.[Medline]
  44. Kartner N., Riordan J., Ling V. Cell surface P-glycoprotein associated with multidrug resistance in mammalian cell lines. Science (Wash. DC), 221: 1285-1288, 1983.[Abstract/Free Full Text]
  45. Fairchild C. R., Ivy S. P., Kao-Shan C-S., Whang-Pen J., Rosen N., Isreal M. A., Melera P. W., Cowan K. H., Goldsmith M. E. Isolation of amplified and overexpressed DNA sequences from Adriamycin-resistant human breast cancer cells. Cancer Res., 47: 5141-5148, 1987.[Abstract/Free Full Text]
  46. Flier J. S., Underhill L. H. Multiple-drug resistance in human cancer. N. Engl. J. Med., 316: 1388-1393, 1987.[Medline]
  47. Endicott J. A., Ling V. The biochemistry of P-glycoprotein-mediated multidrug resistance. Annu. Rev. Biochem., 58: 137-171, 1989.[Medline]
  48. Clarke R., Currier S., Kaplan O., Lovelace E., Boulay V., Gottesman M. M., Dickson R. B. Effect of P-glycoprotein expression on sensitivity to hormones in MCF7 human breast cancer cells. J. Natl. Cancer Inst. (Bethesda), 84: 1506-1512, 1992.[Abstract/Free Full Text]
  49. Gottesman M. M., Pastan I. Biochemistry of multidrug resistance mediated by the multidrug transporter. Annu. Rev. Biochem., 62: 385-427, 1993.[Medline]
  50. Gros P., Croop J., Roninson I., Varshavsky A., Housman D. Isolation and characterization of DNA sequences amplified in multidrug-resistant hamster cells. Proc. Natl. Acad. Sci. USA, 83: 337-341, 1986.[Abstract/Free Full Text]
  51. Roninson I., Chin J., Choi K., Gros P., Housman D., Fojo A., Shen D., Gottesman M., Pastan I. Isolation of human mdr DNA sequences amplified in multidrug-resistant KB carcinoma cells. Proc. Natl. Acad. Sci. USA, 12: 4538-4542, 1986.
  52. Gros P., Neriah Y. B., Croop J., Housman D. Isolation and expression of a complementary DNA that confers multidrug resistance. Nature (Lond.), 323: 728-731, 1986.[Medline]
  53. Combates N. J., Rzepka R. W., Chen Y-N., Cohen D. NFIL-6, a member of the C/EBP family of transcription factors, binds and trans-activates the human MDR1 gene promoter. J. Biol. Chem., 269: 29715-29719, 1994.[Abstract/Free Full Text]
  54. Combates N. J., Kwon P. O., Rzepka R. W., Cohen D. Involvement of the transcription factor NFIL-6 in phorbol ester induction of P-glycoprotein in U937 cells. Cell Growth Differ., 8: 213-219, 1997.[Abstract]
  55. Krylov D., Olive M., Vinson C. Extending dimerization interfaces: the bZIP basic region can form a coiled coil. EMBO J., 14: 5329-5337, 1995.[Medline]
  56. Olive M., Williams S. C., Dezan C., Johnson P. F., Vinson C. Design of a C/EBP-specific, dominant-negative bZIP protein with both inhibitory and gain-of-function properties. J. Biol. Chem., 271: 2040-2047, 1996.[Abstract/Free Full Text]
  57. Lenhert M. Clinical multidrug resistance in cancer: a multifactorial problem. Eur. J. Cancer, 6: 912-920, 1996.
  58. Robert J. Multidrug resistance in oncology: diagnostic and therapeutic approaches. Eur. J. Clin. Investig., 29: 536-545, 1999.[Medline]
  59. Borsellino N., Belldegrun A., Bonavida B. Endogenous interleukin 6 is a resistance factor for cis-diamminedichloroplatinum and etoposide-mediated cytotoxicity of human prostate carcinoma cell lines. Cancer Res., 55: 4633-4639, 1995.[Abstract/Free Full Text]
  60. van Gameren M. M., Willemse P. H. B., Mulder N. H., Limburg P. C., Groen H. J., Vallenga E., de Vries E. G. Effects of recombinant human interleukin-6 in cancer patients: a Phase I-II study. Blood, 84: 1434-1441, 1994.[Abstract/Free Full Text]
  61. Veldhuis G. J., Willemse P. H., Mulder N. H., Limburg P. C., de Vries E. G. Potential use of recombinant human interleukin-6 in clinical oncology. Leuk. Lymphoma, 20: 373-379, 1996.[Medline]
  62. Weber J. S. Clinical trials with IL-6. Ann. N. Y. Acad. Sci., 762: 357-358, 1995.[Medline]
  63. Stouthard J. M., Goey H., de Vries E. G., de Mulder P. H., Groenewegen A., Pronk L., Stoter G., Sauerwein H. P., Bakker P. J., Veenhof C. H. Recombinant human interleukin 6 in metastatic renal cell cancer: a Phase II trial. Br. J. Cancer, 73: 789-793, 1996.[Medline]
  64. Weber J., Gunn H., Yang J., Parkinson D., Topalian S., Schwartzentruber D., Ettinghausen S., Levitt D., Rosenberg S. A. A Phase I trial of intravenous interleukin-6 in patients with advanced cancer. J. Immunother., 15: 292-302, 1994.
  65. Weber J., Yang J. C., Topalian S. L., Parkinson D. R., Schwartzentruber D. S., Ettinghausen S. E., Gunn H., Mixon A., Kim H., Cole D., et al Phase I trial of subcutaneous interleukin-6 in patients with advanced malignancies. J. Clin. Oncol., 11: 499-506, 1993.[Abstract/Free Full Text]
  66. Fojo A. T., Shen D. W., Mickley L. A., Pastan I., Gottesman M. Intrinsic drug resistance in human kidney cancer is associated with expression of human multidrug resistance gene. J. Clin. Oncol., 5: 1922-1927, 1987.[Abstract/Free Full Text]
  67. Kanamaru H., Kakehi Y., Yoshida O., Nakanishi S., Pastan I., Gottesmann M. MDR1 RNA levels in human renal cell carcinomas: correlation with grade and prediction of reversal doxorubicin resistance by quinidine in tumor explants. J. Natl. Cancer Inst. (Bethesda), 81: 844-849, 1989.[Abstract/Free Full Text]
  68. Weinstein R. S., Jakate S. M., Dominguez J. M., Lebovitz M. D., Koukoulis G. K., Kuszak J. R., Klusens L. F., Grogan T. M., Saclarides T. J., Roninson I. B., Coon J. S. Relationship of the expression of the multidrug resistance gene product (P-glycoprotein) in human colon carcinoma to local tumor aggressiveness and lymph node metastasis. Cancer Res., 51: 2720-2726, 1991.[Abstract/Free Full Text]
  69. Chan H. S., DeBoer G., Haddad G., Gallie B. L., Ling V. Multidrug resistance in pediatric malignancies. Hematol. Oncol. Clin. N. Am., 9: 275-318, 1995.[Medline]
  70. Fisher G. A., Lum B. L., Hausdorff J., Sikic B. I. Pharmacological considerations in the modulation of multidrug resistance. Eur. J. Cancer, 6: 1082-1088, 1996.
  71. Leighton J. J., Goldstein L. J. P-glycoprotein in adult solid tumors. Expression and prognostic significance. Hematol. Oncol. Clin. N. Am., 9: 251-273, 1995.[Medline]
  72. Bouffard D., Ohkawa T., Kijima H., Irie A., Suzuki T., Curcio L., Holm P., Sassani A., Scanlon K. Oligonucleotide modulation of multidrug resistance. Eur. J. Cancer, 32A: 1010-1018, 1996.
  73. Ueda K., Clark D., Chen C., Roninson I., Gottesman M., Pastan I. The human multidrug resistance (mdr1) gene. J. Biol. Chem., 262: 505-508, 1987.[Abstract/Free Full Text]
  74. Madden M., Morrow C., Nakagawa M., Goldsmith M., Fairchild C., Cowan K. Identification of 5' and 3' sequences involved in the regulation of transcription of the human mdr1 gene in vivo. J. Biol. Chem., 268: 8290-8297, 1993.[Abstract/Free Full Text]
  75. Bromberg J. F., Wrzeszcynska M. H., Devgan G., Zho Y., Pestell R. G., Albanese C., Darnell J. E. Stat3 as an oncogene. Cell, 98: 295-303, 1999.[Medline]
  76. Qiu Y., Ravi L., Kung H-J. Requirement of ErbB2 for signaling by interleukin-6 in prostate carcinoma cells. Nature (Lond.), 393: 83-85, 1998.[Medline]
  77. Sabbatini A., Basolo F., Valentini P., Mattii L., Calvo S., Fiore L., Ciardello F., Petrini M. Induction of multidrug resistance (MDR) by transfection of MCF-10A cell line with c-Ha-ras and c-erbB2 oncogenes. Int. J. Cancer, 59: 208-211, 1994.[Medline]
  78. Tsai C., Chang K., Perng R., Mitsudomi T., Chen M., Kadoyama C., Gazdar A. Correlation of intrinsic chemoresistance of non-small-cell lung cancer cell lines with HER-2/neu gene expression but not with ras gene mutations. J. Natl. Cancer Inst. (Bethesda), 85: 897-901, 1993.[Abstract/Free Full Text]
  79. Wosikowski K., Schuurhuis D., Kops G., Sceda M., Bates S. Altered gene expression in drug-resistant human breast cancer cells. Clin. Cancer Res., 3: 2405-2414, 1997.[Abstract/Free Full Text]
  80. Dahiya R., Deng G. Molecular prognostic markers in breast cancer. Breast Cancer Res. Treat., 52: 185-200, 1998.[Medline]
  81. Scambia G., Testa U., Benedetti P., Foti E., Martucci R., Gadducci A., Perillo A., Facchini V., Peschle C., Mancuso S. Prognostic significance of interleukin 6 serum levels in patients with ovarian cancer. Br. J. Cancer, 71: 354-356, 1995.[Medline]
  82. Smith P. C., Hobisch A., Lin D. L., Culig Z., Keller E. T. Interleukin-6 and prostate cancer progression. Cytokine Growth Factor Rev., 12: 33-40, 2001.[Medline]
  83. Adler H. L., McCurdy M. A., Kattan M. W., Timme T. L., Scardino P. T., Thompson T. C. Elevated levels of circulating interleukin-6 and transforming growth factor-ß1 in patients with metastatic prostatic carcinoma. J. Urol., 161: 182-187, 1999.[Medline]
  84. Twillie D. A., Eisenberger M. A., Carducci M. A., Hseih W-S., Kim W. Y., Simons J. W. Interleukin-6: a candidate mediator of human prostate cancer morbidity. Urology, 45: 542-549, 1995.[Medline]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
A. DeMichele, R. Gray, M. Horn, J. Chen, R. Aplenc, W. P. Vaughan, and M. S. Tallman
Host Genetic Variants in the Interleukin-6 Promoter Predict Poor Outcome in Patients with Estrogen Receptor-Positive, Node-Positive Breast Cancer
Cancer Res., May 15, 2009; 69(10): 4184 - 4191.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
B. D. Hollingshead, T. V. Beischlag, B. C. DiNatale, P. Ramadoss, and G. H. Perdew
Inflammatory Signaling and Aryl Hydrocarbon Receptor Mediate Synergistic Induction of Interleukin 6 in MCF-7 Cells
Cancer Res., May 15, 2008; 68(10): 3609 - 3617.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
A. K. Sasser, N. J. Sullivan, A. W. Studebaker, L. F. Hendey, A. E. Axel, and B. M. Hall
Interleukin-6 is a potent growth factor for ER-{alpha}-positive human breast cancer
FASEB J, November 1, 2007; 21(13): 3763 - 3770.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. B. Nilsson, G. Armaiz-Pena, R. Takahashi, Y. G. Lin, J. Trevino, Y. Li, N. Jennings, J. Arevalo, S. K. Lutgendorf, G. E. Gallick, et al.
Stress Hormones Regulate Interleukin-6 Expression by Human Ovarian Carcinoma Cells through a Src-dependent Mechanism
J. Biol. Chem., October 12, 2007; 282(41): 29919 - 29926.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
K. M. Hatle, W. Neveu, O. Dienz, S. Rymarchyk, R. Barrantes, S. Hale, N. Farley, K. M. Lounsbury, J. P. Bond, D. Taatjes, et al.
Methylation-Controlled J Protein Promotes c-Jun Degradation To Prevent ABCB1 Transporter Expression
Mol. Cell. Biol., April 15, 2007; 27(8): 2952 - 2966.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
F. Meng, Y. Yamagiwa, S. Taffetani, J. Han, and T. Patel
IL-6 activates serum and glucocorticoid kinase via p38{alpha} mitogen-activated protein kinase pathway
Am J Physiol Cell Physiol, October 1, 2005; 289(4): C971 - C981.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
A. H. Gordon, R. J. O'Keefe, E. M. Schwarz, R. N. Rosier, and J. E. Puzas
Nuclear Factor-{kappa}B-Dependent Mechanisms in Breast Cancer Cells Regulate Tumor Burden and Osteolysis in Bone
Cancer Res., April 15, 2005; 65(8): 3209 - 3217.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
B. Gu, L. Espana, O. Mendez, A. Torregrosa, and A. Sierra
Organ-selective chemoresistance in metastasis from human breast cancer cells: inhibition of apoptosis, genetic variability and microenvironment at the metastatic focus
Carcinogenesis, December 1, 2004; 25(12): 2293 - 2301.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
D. E. Spaner
Amplifying cancer vaccine responses by modifying pathogenic gene programs in tumor cells
J. Leukoc. Biol., August 1, 2004; 76(2): 338 - 351.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
G. K. Chen, S. Sale, T. Tan, R. P. Ermoian, and B. I. Sikic
CCAAT/Enhancer-Binding Protein {beta} (Nuclear Factor for Interleukin 6) Transactivates the Human MDR1 Gene by Interaction with an Inverted CCAAT Box in Human Cancer Cells
Mol. Pharmacol., April 1, 2004; 65(4): 906 - 916.
[Abstract] [Full Text]

Home page
 aacrmtgHome page
A. D. DeMichele, A.-M. Martin, and B. L. Weber
IL6 variants as modifiers of breast cancer outcome.
AACR Meeting Abstracts, March 1, 2004; 2004(1): 1316 - 1317.
[Abstract]

Home page
 Cancer Res.Home page
A. DeMichele, A.-M. Martin, R. Mick, P. Gor, L. Wray, M. Klein-Cabral, G. Athanasiadis, T. Colligan, E. Stadtmauer, and B. Weber
Interleukin-6 -174G->C Polymorphism Is Associated with Improved Outcome in High-Risk Breast Cancer
Cancer Res., November 15, 2003; 63(22): 8051 - 8056.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
S. Diehl, C.-W. Chow, L. Weiss, A. Palmetshofer, T. Twardzik, L. Rounds, E. Serfling, R. J. Davis, J. Anguita, and M. Rincon
Induction of NFATc2 Expression by Interleukin 6 Promotes T Helper Type 2 Differentiation
J. Exp. Med., July 1, 2002; 196(1): 39 - 49.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Conze, D.
Right arrow Articles by Rincón, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Conze, D.
Right arrow Articles by Rincón, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online