MCJ is a unique transmembrane DnaJ protein, highly conserved in vertebrates.
Although some studies have examined the regulation of the MCJ gene by methylation, the biology and function of MCJ protein remain unknown. We therefore performed a PSI-BLAST search (2
) by using the human MCJ sequence to examine the potential evolutionary association of MCJ with other proteins of known functions. The search revealed that MCJ is a member of a set of eukaryotic proteins that contain a conserved (66 to 100%) transmembrane domain and the C-terminal DnaJ domain (Fig. ; see Fig. S1 in the supplemental material). This set includes the previously described yeast TIM14, a component of a mitochondrial inner membrane translocase (42
) (Fig. ). It also includes an uncharacterized human DnaJ protein that has been described as a “translocase of mitochondrion inner membrane” because of its high level of similarity (67%; P
, 4 × 10−15
) to the yeast TIM14. This TIM14 human ortholog, which will be referred to herein as a TIM14-like protein, is similar to human MCJ (74%; P
, 6 × 10−32
) but lacks the corresponding N terminus (Fig. ). The sequence similarity searches indicate no other human proteins within this eukaryotic phylogeny (see Fig. S1 in the supplemental material).
FIG. 1. Phylogenetic analysis and sequence alignment of MCJ. (A) Homologous MCJ sequences from different species found by a BLASTp search. Alignments were made using the T-Coffee program. H. sapiens, Homo sapiens; D. melanogaster, Drosophila melanogaster; A. (more ...)
Five evolutionary clades (i.e., groups of sequences with a common ancestor) (Fig. ) were identified: plant, yeast TIM14, fly-worm TIM14-like, vertebrate TIM14-like, and vertebrate MCJ clades. The plant clade was set as the out-group, meaning that the other four clades share with one another a common ancestor that is not shared with the plant clade (Fig. ). The ecdysozoan (fly and worm) MCJ and TIM14-like sequences form a clade. We found with a high confidence level a gene duplication leading to two vertebrate clades, the MCJ and the TIM14-like clades (Fig. ). Although gene duplication may have occurred after the divergence of vertebrates from the fly-worm (ecdysozoan) lineage (Fig. , upper panel), this possibility was not strongly supported by the results of our analysis (described in Materials and Methods). Stronger support was found for MCJ to be the result of gene duplication prior to the divergence of ecdysozoa from vertebrates (Fig. , lower panel). This scenario would imply that MCJ must then have been lost in the fly-worm lineage(s).
Unlike the transmembrane and C-terminal DnaJ regions that are highly conserved within the five clades, the juxtamembrane C-terminal regions are distinct among clades, although they are conserved within each clade (Fig. ). Thus, vertebrate MCJ orthologs have a unique juxtamembrane C-terminal region that is not conserved in other clades. In addition, the N terminus region (35 aa) present in the MCJ clade is absent in the TIM14-like vertebrate and ecdysozoan clades and is highly variable in the yeast TIM14 clade (Fig. ). However, within the MCJ clade, seven sites of this N terminus are perfectly conserved and more than 90% of the sites exhibit some degree of conservation. The presence of the highly conserved N-terminal region specifically in the MCJ clade, but not in the TIM14-like vertebrate clade, suggests distinct functions of MCJ and TIM14 in vertebrates.
MCJ is a type II transmembrane protein localized in the Golgi compartment.
The above-described MCJ phylogenetic analysis revealed the evolutionary ancestor of MCJ as yeast TIM14, which is localized in the inner mitochondrial membrane (42
). Since no previous studies have addressed the subcellular localization of MCJ, we generated a HA-tagged-MCJ-expressing construct that also contained an internal ribosome entry site-EGFP gene and transfected 293T cells with this construct. We first tested MCJ expression in transfected cells by Western blot analysis. In correlation with the predicted size of MCJ, a protein of approximately 16 to 18 kDa was detected only in MCJ-transfected 293T cells and not in cells transfected with an empty plasmid (Fig. ).
MCJ subcellular localization in transfected cells was examined by confocal microscopy analysis. MCJ was clearly a cytoplasmic protein with a distinct punctate distribution in well-defined areas of the cytosol that resembled the distribution of intracellular organelles (Fig. ). No MCJ staining in the untransfected cells was observed (Fig. ). To investigate whether MCJ was localized in the mitochondria, we costained MCJ-transfected cells with Mitotracker, a specific marker for mitochondria. However, MCJ did not colocalize with the Mitotracker (Fig. ). To test if MCJ was localized in the endoplasmic reticulum, cells were cotransfected with the MCJ-expressing plasmid and the pDsRed2-ER plasmid that expresses a red fluorescence protein targeted to the endoplasmic reticulum by the endoplasmic reticulum retention sequence (KDEL). No clear colocalization of MCJ with the pDsRed2-ER plasmid was observed by confocal microscopy (see Fig. S2 in the supplemental material). To precisely determine the organelle(s) where MCJ was localized, we performed immunoelectron microscopy. MCJ-transfected 293T cells were fixed, embedded, sectioned, and stained with antibody-coated gold particles (pAg10). A clear punctate distribution of MCJ specifically in the Golgi apparatus and other associated vesicles was visualized (Fig. ). No MCJ localization in the mitochondria (Fig. ), endoplasmic reticulum (Fig. ), or nuclear membrane (data not shown) was detected. Thus, MCJ is an intracellular transmembrane protein localized primarily in the Golgi apparatus and associated vesicles.
The superfamily Hidden Markov model protein topology prediction program predicted that MCJ was a type II transmembrane protein (i.e., a protein with an intracellular N terminus and an extracellular C terminus). Since our immunoelectron microscopy studies showed that MCJ was present in the Golgi apparatus, this prediction would suggest that the MCJ C terminus was in the Golgi lumen whereas the N terminus was cytoplasmic. To confirm this prediction and further characterize the orientation of MCJ, we used the permeabilization-semipermeabilization method previously described (39
). This approach is based on comparative epitope accessibilities to the antibody for the detection of intracellular transmembrane protein topology. The classical method of immunostaining involves the permeation of fixed cells with a detergent (e.g., Triton X-100) that allows the antibodies to detect all intracellular proteins independently of their localization patterns. However, the semipermeabilization method uses a rapid freeze-thaw technique that selectively permeates the plasma membrane while the intracellular membranes remain impermeable. Since the HA tag was present at the N terminus of MCJ, we used the anti-HA antibody for the detection of the MCJ N-terminal region. For the detection of the C terminus, we generated a rabbit polyclonal antibody against the C terminus of MCJ. The specificity of this anti-MCJ antibody was examined by Western blot analysis using extracts from HA-MCJ-transfected and untransfected 293T cells. The anti-MCJ antibody was able to detect MCJ only in the MCJ-transfected 293T cells (Fig. ). The specificity of the detected band of MCJ was further demonstrated by reprobing the blot with anti-HA antibody (Fig. ).
We therefore used both anti-HA and anti-MCJ antibodies to detect MCJ by confocal microscopy analysis. 293T cells were transfected with an HA-MCJ-expressing plasmid, fixed, permeabilized, stained with either anti-HA or anti-MCJ antibody, and analyzed by confocal microscopy. Both anti-HA and anti-MCJ antibodies detected MCJ with similar patterns of expression in the transfected cells (Fig. ). In parallel, HA-MCJ-transfected cells were rapidly freeze-thawed for semipermeabilization, fixed, stained with anti-HA and anti-MCJ antibodies, and analyzed by confocal microscopy. MCJ was detected with the anti-HA antibody but not with the anti-MCJ antibody (Fig. ), indicating that the MCJ C terminus was not accessible to the antibody. These results confirm that the N terminus of MCJ is cytosolic whereas the C terminus resides within the Golgi lumen. Thus, MCJ is a type II cochaperone that resides within the Golgi compartment.
MCJ is expressed in drug-sensitive but not in drug-resistant breast cancer cells.
The loss of MCJ expression has been associated with increased resistance to chemotherapeutic drugs in ovarian cancer cells (56
). Multidrug resistance is a common phenomenon observed in other cancer types such as breast cancer. To investigate whether the loss of MCJ expression in drug-resistant cells was extended to cancer types other than ovarian, we examined MCJ expression in breast cancer cells. We compared MCJ expression in drug-sensitive MCF7 breast cancer cells with MCJ expression in MCF7/ADR cells that are derived from MCF7 cells but are resistant to several drugs, including doxorubicin, paclitaxel, and vincristine (18
). Total RNA was isolated from MCF7 and MCF7/ADR cells, and MCJ
expression was examined by conventional RT-PCR using HPRT
as an internal control. MCJ was expressed at high levels in MCF7 cells but was undetectable in MCF7/ADR cells (Fig. ). Similar results were obtained by quantitative real-time RT-PCR analysis of MCJ
FIG. 3. Loss of MCJ gene expression in multidrug-resistant breast cancer cells. (A) Total RNA was extracted from MCF7 and MCF7/ADR breast cancer cells and examined for the expression of MCJ and HPRT genes by RT-PCR. (B) The expression of the MCJ gene relative (more ...)
To confirm the loss of MCJ
expression in multidrug-resistant cells, we used other MCF7-derived cells with a multidrug-resistant phenotype. We have previously shown that multidrug-resistant but not drug-sensitive breast cancer cells produce interleukin-6 (IL-6) and that the stable expression of IL-6 in MCF7 cells confers multidrug resistance (10
). We therefore isolated total RNA from MCF7 cells and MCF7 cells stably expressing IL-6 (MCF7/IL-6 cells) and examined MCJ
expression by RT-PCR. MCJ was expressed in MCF7 cells but not in the multidrug-resistant MCF7/IL-6 cells (Fig. ). We have also shown that the transient treatment of MCF7 cells with exogenous IL-6 increases the drug resistance of these cells (10
). We tested whether this increased resistance was associated with decreased MCJ
expression. MCF7 cells that were treated with exogenous IL-6 for 1 week contained reduced levels of MCJ mRNA compared with untreated MCF7 cells (Fig. ).
To rule out the possibility that the correlation between reduced MCJ expression and chemoresistance was restricted to this breast cancer cell line, we examined another breast cancer cell line, MDA-MB-321, and its doxorubicin-resistant derivative, the MD22 cell line (32
). Unlike the multidrug-resistant MCF7/ADR cells, MD22 cells are resistant specifically to doxorubicin. We isolated RNA and performed RT-PCR to assess MCJ
expression. While MDA-MB-321 cells expressed high levels of MCJ
, very low levels were detected in MD22 cells (Fig. ). To examine whether MCJ expression was also blocked in other multidrug-resistant cells, we examined the drug-sensitive MES-SA uterine cancer cell line and its multidrug-resistant derivative, the MES-SA/Dx5 cell line (27
), by RT-PCR. As in MCF7 cells, MCJ
was expressed in the MES-SA cells, but its expression was undetectable in MES-SA/Dx5 cells (Fig. ). Thus, the loss of MCJ gene expression in drug-resistant cells may be a common phenomenon in a number of solid tumors, suggesting that MCJ can be a multidrug resistance marker independent of the cancer type.
Since the sensitivity of the rabbit anti-MCJ polyclonal antibody was limited for the detection of endogenous MCJ (data not shown), we generated a monoclonal antibody against the N-terminal peptide (aa 1 to 35) of MCJ that has no homology with corresponding regions of other mammalian proteins, as described above (Fig. ). To first test the specificity of the anti-MCJ mAb and its ability to recognize MCJ, we performed Western blot analysis using whole-cell extracts from MCJ-transfected and untransfected 293T cells. A unique band of the corresponding size was detected exclusively in MCJ-transfected cells (Fig. ). To further confirm the specificity of the anti-MCJ mAb, we performed confocal microscopy with MCJ-transfected and untransfected 293T cells. Anti-MCJ reactivity was detected only in the transfected and not in the untransfected cells (Fig. ), and the pattern of expression was similar to that observed with the antitag and the polyclonal anti-MCJ antibodies (Fig. ).
FIG. 4. Loss of MCJ in multidrug-resistant breast cancer cells. (A) 293T cells were transfected with an MCJ-expressing plasmid (MCJ) or a control plasmid (Con) and lysed, and MCJ expression was determined by Western blot analysis using an anti-MCJ mAb (MCJ). (more ...)
We used the anti-MCJ mAb to further demonstrate the selective expression of MCJ in drug-sensitive breast cancer cells but not in drug-resistant cells by Western blot analysis. A distinct band of a size corresponding to MCJ was present in MCF7 cells but not in multidrug-resistant MCF7/ADR and MCF7/IL-6 cells (Fig. ). No other cross-reactive proteins in MCF7 cells could be detected with this mAb. In addition, MCJ was present in the drug-sensitive MDA-MB-231 breast cancer cells while only very low MCJ levels were detected in the doxorubicin-resistant MD22 cells (Fig. ). Thus, the expression of MCJ correlated with the MCJ mRNA levels detected in these cells, further demonstrating the loss of MCJ in drug-resistant breast cancer cells.
We also investigated the subcellular distribution of endogenous MCJ in MCF7 cells by immunostaining and confocal microscopy using the anti-MCJ mAb. Although lower than that in MCJ-overexpressing 293T cells, the level of expression of endogenous MCJ in MCF7 cells was clearly detectable (Fig. ). No MCJ could be detected in MCF7/ADR cells (Fig. ). Endogenous MCJ also showed a punctate distribution pattern in MCF7 cells, although it was more widely distributed through the cytoplasm. Several studies have shown that the Golgi compartments of MCF7 cells are diffusely localized throughout the cytoplasm (5
). We examined the colocalization of MCJ with the human trans-Golgi network protein TGN46 that has been previously used to detect the Golgi network in MCF7 cells (59
). Confocal microscopy analysis showed that MCJ partially colocalized with TGN46 (Fig. ). Together, these results indicate that endogenous MCJ is localized in the Golgi compartment in drug-sensitive breast cancer cells but its expression is lost in multidrug-resistant cells.
MCJ is required for breast cancer cells to maintain the response to chemotherapeutic drugs.
To address whether the absence of this Golgi compartment-associated protein by itself could induce multidrug resistance in breast cancer cells, we examined the effect of the inhibition of MCJ expression by RNA interference. An siRNA MCJ target sequence (siMCJ) was cloned downstream of the H1 RNA polymerase III promoter in pSuperEGFP, a modified version of the pSuper plasmid (6
) that includes the EGFP gene the under control of the CMV promoter. MCF7 cells were transiently transfected with pSuperEGFP-siMCJ or an empty plasmid. Cells transfected with siMCJ had lower levels of MCJ mRNA than cells transfected with the empty plasmid, as determined by RT-PCR (Fig. ). We then performed the stable transfection of MCF7 cells with the pSuperEGFP-siMCJ construct, generating MCF7/siMCJ cells. Two clones, MCF7/siMCJ-1B and MCF7/siMCJ-3B, were selected for further expansion and characterization. Total RNA was isolated from MCF7, MCF7/siMCJ-1B, and MCF7/siMCJ-3B cells and examined for MCJ gene expression. No MCJ mRNA was detected in MCF7/siMCJ-3B and MCF7/siMCJ-1B cells by conventional RT-PCR (Fig. ), and very low levels were detected only in MCF7/siMCJ-1B cells by quantitative real-time RT-PCR (Fig. ).
FIG. 5. MCJ is required for breast cancer cells to maintain a chemotherapy response. (A) MCF7 cells were transfected with the empty pSuperEGFP (control) or pSuperEGFP-siMCJ (siMCJ) plasmid. Total RNA was extracted 36 h after transfection, and MCJ (M) and HPRT (more ...)
To confirm that the expression of siMCJ in MCF7/siMCJ cells abolishes not only MCJ mRNA expression but also protein expression, we examined endogenous MCJ levels by Western blotting using the anti-MCJ mAb. MCJ was clearly present in MCF7 cells, but it was almost undetectable in MCF7/siMCJ-1B and MCF7/siMCJ-3B cells, as well as in MCF7/ADR cells (Fig. ) These data indicate that MCJ mRNA and protein expression were abolished in MCF7/siMCJ cells.
The MCF7/siMCJ-1B and -3B cells had a rate of proliferation similar to that of MCF7 cells, and no difference in the viability of these cells in culture was observed (data not shown). We examined whether the inhibition of MCJ expression could increase resistance to doxorubicin (anthracycline), a commonly used chemotherapeutic drug for breast cancer treatment. MCF7, MCF7/ADR, MCF7/siMCJ-1B, and MCF7/siMCJ-3B cells were cultured in the absence or presence of different concentrations of doxorubicin and the percent viability was measured by the MTT assay. In correlation with results from previous studies (1
), MCF7 cells were highly sensitive to doxorubicin while MCF7/ADR cells were highly resistant (Fig. ). Interestingly, MCF7/siMCJ-1B and MCF7/siMCJ-3B cells were significantly more resistant to doxorubicin than MCF7 cells (Fig. ).
We also tested the response to paclitaxel (taxane), another commonly used chemotherapeutic drug for breast cancer. MCF7 cells were highly sensitive to paclitaxel (Fig. ). In contrast, MCF7/siMCJ-1B and MCF7/siMCJ-3B cells were highly resistant, similar to MCF7/ADR cells (Fig. ). Together, these results demonstrate that MCJ is required for breast cancer cells to respond to different drugs such as doxorubicin and paclitaxel and that the inhibition of MCJ expression causes multidrug resistance.
MCJ is required for the intracellular accumulation of chemotherapeutic drugs.
Impaired intracellular accumulation of chemotherapeutic drugs due to transport-mediated efflux is the best-characterized mechanism involved in multidrug resistance (38
). We examined the effect of MCJ downregulation on the intracellular accumulation of doxorubicin by confocal microscopy. MCF7, MCF7/siMCJ-1B, and MCF7/siMCJ-3B cells were treated with medium alone or with doxorubicin for 1, 2, or 3 h. Cells were then washed and fixed, and doxorubicin fluorescence was visualized by confocal microscopy. No doxorubicin fluorescence was detected in MCF7 cells treated with medium alone or with doxorubicin for only 1 h (data not shown). After 2 h of treatment, some doxorubicin fluorescence was detected in the MCF7 cells, but the maximum level of intracellular accumulation was reached after 3 h (Fig. ). Both MCF7/siMCJ-1B and MCF7/siMCJ-3B cells expressed EGFP, but no doxorubicin fluorescence was observed in these cells after 3 h of treatment (Fig. ). Similarly, no doxorubicin fluorescence in MCF7/siMCJ cells was detected after shorter (1- and 2-h) and longer (4-h) periods of treatment (data not shown).
FIG. 6. MCJ is required for the intracellular accumulation of doxorubicin. (A) MCF7, MCF7/siMCJ-1B, and MCF7/siMCJ-3B cells were incubated in the absence (medium) or the presence (Dox) of doxorubicin (3 μM) for the indicated periods of time. Doxorubicin (more ...)
To demonstrate that this phenotype was due to the inhibition of MCJ expression rather than the selection of the siMCJ cell clones, MCF7 cells were transiently transfected with either an empty pSuperEGFP plasmid or the pSuperEGFP-siMCJ plasmid. Thirty-six hours after transfection, cells were treated with doxorubicin for 3 h and examined by confocal microscopy analysis. Transfected cells were identified by the presence of EGFP. Both EGFP-positive and EGFP-negative cells among the control plasmid-transfected MCF7 cells showed doxorubicin accumulation (Fig. ). In contrast, doxorubicin fluorescence could be detected only in EGFP-negative, not EGFP-positive, siMCJ-transfected MCF7 cells (Fig. ). Thus, the transient inhibition of MCJ expression interferes with the intracellular accumulation of the drug.
To confirm the confocal microscopy results, we examined the intracellular accumulation of doxorubicin by flow cytometry. MCF7, MCF7/ADR, MCF7/siMCJ-1B, and MCF7/siMCJ-3B cells were treated with doxorubicin (0.3 and 3 μM) for 3 h, washed extensively, and examined by flow cytometry. High levels of doxorubicin were present in MCF7 cells even at the lower dose (Fig. ). In contrast, no intracellular accumulation of doxorubicin could be detected in MCF7/siMCJ-1B and -3B cells at the lower dose of doxorubicin (0.3 μM) and very low intracellular levels were detected at the higher dose (3 μM) (Fig. ). In addition, no doxorubicin in MCF7/ADR cells was observed (Fig. ). The intracellular accumulation of doxorubicin was also impaired in other cells that have lost MCJ, including MCF7/IL-6 and MES/DOX cells (Fig. S3 in the supplemental material) Together, these results demonstrate that the presence of MCJ is required to allow the intracellular accumulation of the drug.
MCJ suppresses ABCB1 gene expression.
Specific ATP-binding cassette (ABC) transporters that promote drug efflux or drug retention in intracellular compartments of cancer cells provide one of the mechanisms to prevent drugs from reaching their specific intracellular targets. The ABC transporters constitute a large family, with 48 members in humans. Some ABC transporters that use specific drugs as substrates are overexpressed in the cancer cell lines and tumors that are multidrug resistant (24
). The substrates of a large number of these transporters, however, remain unknown. The best-characterized member of this family is ABCB1 (also known as the mdr1
or P-glycoprotein). Since ABCB1 is known to be absent in drug-sensitive MCF7 cells (19
), we examined its expression in MCF7/siMCJ cells by Western blot analysis. In contrast to the lack of ABCB1 in MCF7 cells, high levels of ABCB1 were present in MCF7/siMCJ-1B and -3B cells (Fig. ). As previously described, high levels of ABCB1 were also present in MCF7/ADR cells (Fig. ).
FIG. 7. MCJ suppresses ABCB1 gene expression. (A) Whole-cell extracts from MCF7 (M), MCF7/ADR (ADR), and MCF7/siMCJ (siMCJ)-1B and -3B cells were used to examine ABCB1 expression by Western blot analysis. Actin was also examined as a loading control. (B) Total (more ...)
To determine whether the effect of MCJ on ABCB1 levels could be due to changes in the expression of the ABCB1 gene, we measured ABCB1 mRNA levels by conventional RT-PCR. The ABCB1 gene was not expressed in MCF7 cells (Fig. ), but it was highly expressed in MCF7/siMCJ-1B and -3B cells (Fig. ). Similar results were obtained by quantitative real-time RT-PCR (Fig. ). In addition, the inhibition of MCJ expression in MCF7 cells by transient transfection with the pSuperEGFP-siMCJ plasmid caused an upregulation of ABCB1 gene expression (data not shown). Unlike that of ABCB1, the expression of other multidrug ABC transporters like ABCC1
(for multidrug resistance-associated protein) (15
) and ABCG2
(for breast cancer resistance protein) (16
) that are expressed in MCF7 cells was not altered in MCF7/siMCJ cells (Fig. ). Thus, MCJ appears to selectively regulate ABCB1 gene expression.
To further demonstrate the negative role of MCJ in ABCB1 gene expression, we examined whether the expression of MCJ in MCF7/ADR cells downregulates ABCB1 expression. MCF7/ADR cells were transfected with an MCJ-expressing plasmid. Stably MCJ-transfected MCF7/ADR clones (MCF7/ADR-MCJ) were selected. The expression of MCJ did not affect the survival or the proliferation of these cells (data not shown). We examined the ABCB1 expression in these cells by Western blot analysis. Although not totally abolished, the ABCB1 levels in MCF7/ADR-MCJ were reduced compared with those in MCF7/ADR cells (Fig. ). Consistent with the reduced levels of ABCB1, MCF7/ADR-MCJ cells also showed increased intracellular doxorubicin accumulation compared with the parental MCF7/ADR cells (see Fig. S3 in the supplemental material) Together, these results indicate that MCJ is able to negatively regulate ABCB1 expression.
Multidrug resistance induced by the loss of MCJ expression is mediated by ABCB1.
To determine whether the presence of ABCB1 expression in MCF7/siMCJ cells was responsible for the inability of these cells to accumulate doxorubicin, we examined the effect of verapamil, a known pharmacological inhibitor of ABCB1 (8
). MCF7/siMCJ-1B and MCF7/ADR cells were treated with doxorubicin for 3 h in the presence or absence of verapamil. Intracellular drug accumulation was examined by confocal microscopy. No doxorubicin fluorescence in MCF7/siMCJ-1B cells was detected, but clear intracellular accumulation after the addition of verapamil was observed (Fig. ). Similar results with MCF7/ADR cells were observed (Fig. ). To confirm these results, we measured doxorubicin fluorescence by flow cytometry analysis. Verapamil allowed the intracellular accumulation of doxorubicin in MCF7/siMCJ-1B cells and had no effect on MCF7 cells (Fig. ).
FIG. 8. Multidrug resistance induced by the loss of MCJ expression is mediated by ABCB1. (A) MCF7/ADR and MCF7/siMCJ-1B cells (green) were treated with doxorubicin (3 μM) for 3 h in the absence (Dox) or the presence (Dox + Verap.) of the ABCB1 (more ...)
In contrast to anthracyclines and taxanes, 5-FU is not a substrate for ABCB1, and MCF7/ADR cells are therefore as sensitive to this drug as MCF7 cells (41
). To further confirm the involvement of ABCB1 in the multidrug resistance of MCF7/siMCJ cells, we examined the response of these cells to 5-FU by using the MTT assay. The responses of MCF7, MCF7/ADR, and MCF7/siMCJ-1B and -3B cells to doses of 5-FU were comparable (Fig. ). Thus, the loss of MCJ confers resistance to specific drugs that have been associated with the ABCB1 transporter. To further confirm the contribution of ABCB1 to the multidrug resistance induced by the loss of MCJ, we examined the effect of the inhibition of ABCB1 expression by siRNA on intracellular doxorubicin accumulation. MCF7/siMCJ cells were transiently transfected with siRNA oligonucleotides targeting ABCB1, and after 48 h cells were treated with doxorubicin. The intracellular accumulation of doxorubicin was measured by flow cytometry. We observed increased intracellular levels of doxorubicin in MCF7/siMCJ cells transfected with ABCB1-targeting siRNA oligonucleotides (Fig. ). No effect on drug accumulation was detected when scrambled siRNA oligonucleotides or siRNA oligonucleotides corresponding to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were used as controls (Fig. S4 in the supplemental material). The partial effect of ABCB1 siRNA on drug accumulation correlated with an only partial decrease of ABCB1 levels as determined by Western blot analysis (Fig. ). Together, these results indicate that the inability of MCF7/siMCJ cells to accumulate doxorubicin is at least partially mediated by the upregulation of ABCB1, although we do not discard a potential contribution of other ABC transporters with unknown functions.
The absence of MCJ increases c-Jun levels and transcriptional activity.
Several transcription factors have been shown to be involved in the regulation of ABCB1 gene expression, including AP-1, C/EBP, and NF-κB (55
). To investigate the mechanism by which MCJ regulates ABCB1 expression, we examined AP-1, C/EBP, and NF-κB DNA binding by EMSA by using the nuclear extracts from MCF7 and MCF7/siMCJ cells. No difference in levels of C/EBP DNA binding between MCF7 and MCF7/siMCJ cells was observed (Fig. ). Low levels of NF-κB DNA binding in MCF7 cells could be detected, and the levels in MCF7/siMCJ cells were practically undetected (Fig. ). In contrast, the level of AP-1 DNA binding in MCF7/siMCJ cells was greatly increased compared with that in MCF7 cells (Fig. ).
FIG. 9. MCJ downregulates ABCB1 expression by modulation of the AP-1 transcription factor. (A) Nuclear extracts from MCF7 (M) and MCF7/siMCJ (siMCJ)-1B and -3B cells were examined by EMSA using 32P-labeled double-stranded oligonucleotides specific for the C/EBP, (more ...)
AP-1 is composed of either heterodimers of Jun and Fos family members or homodimers of Jun family members (25
). To identify the composition of the AP-1 complex in the MCF7/siMCJ cells, we performed supershift analysis with antibodies specific for AP-1 components by using nuclear extracts from these cells. Anti-JunB and anti-Fos antibodies did not substantially compete with AP-1 DNA binding, and no supershift complex could be detected, indicating that neither of these members was present in the complex (Fig. ). In contrast, anti-c-Jun antibody strongly inhibited the AP-1 binding and a supershift complex was present (Fig. ). Similarly, an antibody that does not supershift but competes with the DNA binding of the three Jun family members (c-Jun, JunB, and JunD) also inhibited the AP-1 complex present in the MCF7/siMCJ cells (Fig. ). These results indicate that the AP-1 complexes present in MCF7/siMCJ cells consist predominantly of c-Jun dimers.
To examine whether increased c-Jun DNA binding resulted in increased AP-1-mediated transcription, we transfected MCF7 and MCF7/siMCJ cells with an AP-1-luciferase reporter construct. In correlation with the high level of AP-1 DNA binding activity, increased AP-1 transcriptional activity was detected in MCF7/siMCJ cells (Fig. ). The loss of MCJ therefore induced AP-1-mediated transcription.
We determined whether the increased c-Jun DNA binding in MCF7/siMCJ cells could be due to an upregulation of c-Jun protein levels. We examined the levels of c-Jun by Western blot analysis using whole-cell lysates. Very low levels of c-Jun were detected in MCF7 cells, but high levels were present in MCF7/siMCJ cells (Fig. ), as well as in MCF7/ADR cells (Fig. ). In contrast, no significant difference in c-Jun mRNA levels was observed by quantitative real-time PCR (Fig. ), suggesting that MCJ may have an effect on c-Jun protein stability or synthesis. It has been previously reported that c-Jun levels can be regulated by ubiquitination and proteasome-mediated degradation (62
). We examined whether the low levels of c-Jun in MCF7 cells were due to increased proteasomal degradation by treating these cells with the proteasome inhibitor MG132 and performing Western blot analysis. A remarkable increase in the levels of c-Jun in the MG132-treated MCF7 cells compared with those in untreated MCF7 cells was observed (Fig. ). MG132 treatment did not increase c-Jun protein levels in MCF7/siMCJ cells (Fig. ). Thus, the lower levels of c-Jun present in MCF7 cells than in MCF7/siMCJ cells are likely due to an increased rate of degradation of c-Jun in the former cells.
A functional AP-1 binding site within the ABCB1 promoter region that binds c-Jun dimers has been described, and a number of studies have shown the regulation of ABCB1
by c-Jun in multidrug-resistant cancer cells (12
). To show that c-Jun is responsible for the induction of ABCB1 gene expression in MCF7/siMCJ cells, we inhibited c-Jun-mediated transcription. The phosphorylation of c-Jun at Ser-63 and Ser-73 by JNK leads to the activation of c-Jun (14
). We transiently transfected MCF7/siMCJ cells with a dominant negative JNK1 (dnJNK1) mutant-expressing plasmid (14
) to inhibit c-Jun activation. ABCB1 expression in untransfected and transfected cells was examined by Western blotting. The presence of dnJNK1 in MCF7/siMCJ-1B and -3B cells caused a substantial reduction of the ABCB1 levels (Fig. ). Thus, the induction of ABCB1 expression in the absence of MCJ required the c-Jun/JNK pathway. A reduction of the ABCB1 levels by the expression of dnJNK1 in MCF7/ADR cells was also observed (see Fig. S5A in the supplemental material).
In addition to the transcription activity, the phosphorylation of c-Jun by JNK has been shown to protect c-Jun from ubiquitination and degradation (22
). We therefore examined the levels of c-Jun in MCF7/siMCJ cells transfected with the dnJNK1 mutant. In correlation with the reduction of ABCB1 levels, the levels of c-Jun were substantially decreased in the presence of dnJNK1 (Fig. ). Similar results were obtained with MCF7/ADR cells (Fig. S5A in the supplemental material), but the expression of dnJNK did not affect the low c-Jun levels present in MCF7 cells (see Fig. S5B in the supplemental material). The amount of total JNK1/JNK2 as determined by Western blot analysis was not increased by the loss of MCJ in MCF7/siMCJ cells (see Fig. S5C in the supplemental material). Although the basal levels of phosphorylated JNK seemed to be slightly increased in both MCF7/siMCJ and MCF7/ADR cells compared with those in MCF7 cells, they were very low since a long film exposure was needed for detection by Western blot analysis (see Fig. S5C in the supplemental material).
Together, these results indicate that the presence of MCJ in MCF7 cells prevents the accumulation of c-Jun, probably by promoting its degradation, and that this process is impaired by the loss of MCJ, suggesting that MCJ may physically interact with c-Jun in MCF7 cells. To address this hypothesis, we performed coimmunoprecipitation analysis of MCJ with c-Jun. Since the levels of c-Jun in MCF7 cells are very low, we used whole-cell extracts from untreated MCF7 cells and from MCF7 cells treated for a short period of time with the proteosome inhibitor MG132 to allow the accumulation of c-Jun. MCJ was immunoprecipitated from whole-cell lysates generated from MCF7 cells or MG132-treated MCF7 cells. Immunoprecipitates were then analyzed for the presence of c-Jun by Western blot analysis. Low but detectable levels of c-Jun coimmunoprecipated with MCJ from MCF7 cells, and higher levels coimmunoprecipitated with MCJ from the MG132-treated cells (Fig. ). Thus, MCJ associates with c-Jun and this association likely favors c-Jun degradation.