Regulation of Mcl-1 by constitutive activation of NF-kappaB contributes to cell viability in human esophageal squamous cell carcinoma cells

Background Esophageal squamous cell carcinoma (ESCC) is one of the most lethal malignancies with a 5-year survival rate less than 15%. Understanding of the molecular mechanisms involved in the pathogenesis of ESCC becomes critical to develop more effective treatments. Methods Mcl-1 expression was measured by reverse transcription (RT)-PCR and Western blotting. Human Mcl-1 promoter activity was evaluated by reporter gene assay. The interactions between DNA and transcription factors were confirmed by electrophoretic mobility shift assay (EMSA) in vitro and by chromatin immunoprecipitation (ChIP) assay in cells. Results Four human ESCC cell lines, TE-1, Eca109, KYSE150 and KYSE510, are revealed increased levels of Mcl-1 mRNA and protein compare with HaCaT, an immortal non-tumorigenic cell line. Results of reporter gene assays demonstrate that human Mcl-1 promoter activity is decreased by mutation of kappaB binding site, specific NF-kappaB inhibitor Bay11-7082 or dominant inhibitory molecule DNMIkappaBalpha in TE-1 and KYSE150 cell lines. Mcl-1 protein level is also attenuated by Bay11-7082 treatment or co-transfection of DNMIkappaBalpha in TE-1 and KYSE150 cells. EMSA results indicate that NF-kappaB subunits p50 and p65 bind to human Mcl-1-kappaB probe in vitro. ChIP assay further confirm p50 and p65 directly bind to human Mcl-1 promoter in intact cells, by which regulates Mcl-1 expression and contributes to the viability of TE-1 cells. Conclusions Our data provided evidence that one of the mechanisms of Mcl-1 expression in human ESCC is regulated by the activation of NF-kappaB signaling. The newly identified mechanism might provide a scientific basis for developing effective approaches to treatment human ESCC.


Background
Human esophageal squamous cell carcinoma (ESCC) is one of the most frequently diagnosed carcinomas, ranked as the sixth leading cause of death from cancers worldwide. ESCC remains the most common histology and occurs at a very high frequency in China, South Africa, France and Italy [1]. Although modest advances have been made in chemotherapy for esophageal cancer, ESCC is still one of the most aggressive types of cancer with a 5-year survival rate less than 15%. The underlying reasons for this disappointingly low survival rate remains to be greatly elucidated. Therefore, a better understanding of the molecular mechanisms of ESCC pathogenesis is expected to facilitate the development of novel therapies for this disease.
The Mcl-1 is an antiapoptotic gene of the Bcl-2 family members. Mcl-1 is overexpressed in many human tumor specimens, including hepatocellular carcinoma [2], pancreatic cancer [3], prostate cancer [4] and others [5]. Overexpression of Mcl-1 was found in malignant melanoma compared to benign nevi and increased expression of Mcl-1 was also observed by comparing primary and metastatic melanoma samples utilizing a tissue microarray [6]. In addition, frequent Mcl-1 gene amplification was identified in lung, breast, neural and gastrointestinal cancers, through which cancer cells depend on the expression of this gene for survival [7]. A survey of antiapoptotic Bcl-2 family member expression in breast, brain, colon, lung, ovarian, renal and melanoma cell lines revealed that Mcl-1 mRNA is more abundant than Bcl-2 or Bcl-xL [8]. These studies demonstrated that Mcl-1 plays a critical role in carcinogenesis and malignancy development in a broad range of human tumors, making it an attractive therapeutic target. However, the underlying mechanisms causing its elevation are not fully understood.
Expression of Mcl-1 gene can be regulated at transcriptional level. Analysis of human Mcl-1 gene 5′-flanking promoter regions for potential transcription factor binding sites revealed consensus sequences including STAT, SRE, Ets, Sp1, CRE-BP [9]. Multiple intracellular signaling pathways and transcription factors have been confirmed to influence Mcl-1 expression, including PI3K/ Akt [10], Stat3 [11,12], CREB [10], Ets family members Elk-1 [13] and PU.1 [14]. In addition, putative binding sites for NF-κB were identified in the Mcl-1 promoter region [9]. Previous studies demonstrated that inhibition of NF-κB activation by a novel NF-κB inhibitor V1810 [15] or Thiocolchicoside [16] accompanied by the downregulation of Mcl-1 expression. However, the underlying mechanistic link between NF-κB and Mcl-1 expression has not been clearly established in these studies. Moreover, although reports [17,18] have revealed that p65 subunit of NF-κB involves in TRAIL induced expression of Mcl-1 in HCT-116 colon carcinoma cells [17] and the interaction of p65 with N-a-Acetyltransferase 10 protein regulates Mcl-1 expression [18], the precise mechanism of Mcl-1 transcriptionally controlled by NF-κB family members is not fully elucidated. Therefore, a better understanding the role of this regulatory molecule in Mcl-1 expression in cancers may allow for the development of rational therapeutics that control Mcl-1 levels.
Transcripition factor NF-κB comprised of homo-and heterodimers of the RelA (p65), RelB, c-Rel, p50/p105 (NF-κB1) and p52/p100 (NF-κB2) polypeptides can both induce and repress gene expression by binding to discrete κB elements in promoters and enhancers. The genes regulated by NF-κB include those controlling apoptosis, cell adhesion, proliferation, and inflammation. In most untransformed cell types, NF-κB complexes are largely cytoplasmic by a family of inhibitory proteins known as inhibitors of NF-κB (IκBs) and therefore remain transcriptionally inactive [19]. Activation of NF-κB typically involves the phosphorylation of IκB by the IκB kinase (IKK) complex, which results in IκB degradation. This liberates NF-κB and allows it to translocate freely to the nucleus and binds to the κB elements in the relevant downstream genes to activate a series of transcriptional events [19]. It has become apparent that aberrant activation of NF-κB in human cancers are common [20]. Activation of NF-κB has been detected in tumor samples from patients, such as breast, colorectal, ovarian, pancreatic, prostate cancers and so forth [21,22]. Constitutive NF-κB activation has also reported in esophageal carcinoma tissues [22,23] and cell lines [24], implying NF-κB activation plays an important role in the tumorigenesis and development of human ESCC. Expression of Mcl-1 has been shown in human esophageal carcinoma cell lines CE81T/VGH [25] and KYSE450 [26]. We thus speculated that a direct link might exist between NF-κB and Mcl-1 expression in human ESCC.
The present study was performed to determine whether Mcl-1 expression is modulated by NF-κB signal pathway in human ESCC. Using human ESCC cell lines as models, reporter gene assays demonstrate that human Mcl-1 promoter activity is decreased by mutation of κB site, specific NF-κB inhibitor Bay11-7082 or dominant inhibitory molecule DNMIκBα in TE-1 and KYSE150 cells. Mcl-1 level is attenuated by Bay11-7082 treatment or co-transfection of DNMIκBα in TE-1 and KYSE150 cells. NF-κB subunits p50 and p65 are further confirmed bound to Mcl-1-κB probe in vitro by EMSA assay and directly bound to human Mcl-1 promoter in intact cells by ChIP assay, respectively. Our data provided evidence that one of the regulatory mechanisms by which Mcl-1 expression in human ESCC is by binding of p50 and p65 to κB site within human Mcl-1 promoter. This NF-κB mediating Mcl-1 expression also contributes to the viability of TE-1 cells. In conclusion, the newly identified mechanism might provide a scientific basis for developing effective approaches to treatment human ESCC.

Cell lines and culture
Human esophageal carcinoma cell lines TE-1 and Eca109 were purchased from Cell Bank of Chinese Academy of Sciences, Shanghai, China. Human esophageal carcinoma cell lines KYSE150 and KYSE510 were kindly provided by Dr. Qian Tao from The Chinese University of Hong Kong, HongKong, China. Immortalized human keratinocyte cell line HaCaT derived from human adult trunk skin was previous described [27,28]. TE-1, Eca109, KYSE150 and KYSE510 cells were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 100 mg/ml streptomycin. HaCaT was cultured in DMEM medium (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum and antibiotics as described above. All cell lines were incubated at 37˚C in a humidified atmosphere containing 5% CO2.

Chemicals and cell treatments
The specific NF-κB inhibitor Bay11-7082 (Calbiochem, Darmstadt, Germany) was prepared as a stock solution of 20 mM in DMSO (Sigma, St. Louis, MO). Subconfluent cells were treated with the compound at indicated concentrations for an indicated time. Detailed treatment procedures were described in figure legends. The final concentration of DMSO in the culture media was kept less than 0.1% which had no significant effect on the cell growth. Vehicle controls were prepared for all treatments.

Transfection and luciferase reporter assays
Cells were cultured in 24-well plates at a density of 1 × 10 5 per well overnight and transfected with Lipofecta-mine™ 2000 (Invitrogen, Carlsbad, CA) according to manufacturer's instructions. In luciferase assay for NF-κB transactivation, each transfection contained 800 ng/ well of pGL3-Basic or pGL3-NF-κB-Luc together with 40 ng/well of internal control pRL-SV40 (Promega, Madison, WI) (Total DNA 840 ng/well). 24 h after transfection, cells were either left untreated (DMSO) or treated with 20 μM Bay11-7082 for 12 h. Cells were harvested at 36 h after transfection and lysates were analyzed for luciferase activity using the Dual Luciferase Reporter assay (Promega, Madison, WI) with a GloMax™ Microplate Luminometer (Promega, Madison, WI). In luciferase assay for the Mcl-1 promoter, each transfection contained 400 ng/well of pGL2-Basic, pGL2-Mcl-1-κBwt or pGL2-Mcl-1-κBmt together with 400 ng/well of pcDNA3.1 or pcDNA3-DNMIκBα expression plasmid. Each transfection contained 40 ng/well of pRL-SV40 as internal control (Total DNA 840 ng/well). 24 h after transfection, cells were either left untreated (DMSO) or treated with 20 μM Bay11-7082 for 12 h. Cells were harvested at 36 h after transfection and lysates were analyzed as described above. The pRL-SV40 was co-transfected in all experiments to correct the variations in transfection efficiency. The data represent the mean ± S.D. of at least two independent experiments performed in triplicate.

RNA interference
TE-1 cells were grown in 6-well plates at a density of 3 × 10 5 cells per well overnight. Cells reached 60-70% confluency on the day of transfection and were transfected with a p50 (sc-29407; 100 pmol), a p65 (sc-29410; 100 pmol) or a scrambled control (sc-37007; 100 pmol) siRNA (all from Santa Cruz Biotechnology) using HiPer-Fect transfection reagent (Cat no: 301705, Qiagen) for 72 h according to the manufacturer's instructions. Cells were harvested for protein extraction and immunoblotting to confirm p50 or p65 knockdown.

Cell viability assay
Cell viability assays were performed using the 4-[3-(4iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assay kit (Roche, Indianapolis, IN) according to the manufacturer's instructions. The assay is based on the cleavage of WST-1 to formazan dye by cellular mitochondrial dehydrogenases. Because cleavage of WST-1 to formazan dye occurs only in viable cells, the amount of dye produced, measured in OD values, directly corresponds with the number of viable cells present in the culture. Briefly, TE-1 cells were firstly transfected with the control, p50 or p65 siRNA in sixwell plates as described above. To investigate whether reintroduction of Mcl-1 restored cell viability, 24 h following the first transfection, a second transient transfection was carried out to ectopically express Mcl-1. Each transfection contained 2 μg pCMV6-A-Puro empty vector or pCMV6-A-Puro-Mcl construct using SuperFect transfection reagent (Cat no: 301305, Qiagen) according to the manufacturer's instructions. At 24 h posttransfection, cells were trypsinized, an aliquot of cells was maintained in six-well plate, harvested at 120 h after NF-κB subunit siRNA transfection and analyzed the Mcl-1 levels by Western blotting. The remainder was transferred as six replicates to 96-well plates at a concentration of 2.5 × 10 3 cells per well in 100 μl of complete RPMI 1640. After culturing for another 24, 48, 72 h (i.e. 72 h, 96 h, 120 h after each siRNA transfection, respectively), 10 μl of WST-1 was added to each well and cells incubated for 2 h at 37°C. The cellular reduction of WST-1 to formazan and its absorbance were measured at 450 nm.

Statistical analysis
Statistical analysis was done with the statistical software program SPSS ver.12.0. Results expressed as mean ± S.D. were analyzed using the Student's t test. Differences were considered significant when P value was <0.05.

Expression of Mcl-1 mRNA and protein in human esophageal squamous cell carcinoma cell lines
To investigate the expression patterns of Mcl-1 in human ESCC cell lines, Mcl-1 expression was first measured by Western blotting. As shown in Figure 1A, four human esophageal carcinoma cell lines, including TE-1, Eca109, KYSE150 and KYSE510 revealed increased levels of Mcl-1 protein compare with an immortal non-tumorigenic keratinocyte HaCaT cell line [27], which was used as a normal control [36,37] for Mcl-1 expression. The Mcl-1 protein levels among these esophageal carcinoma cell lines were similar ( Figure 1A). In addition, semi-quantitative RT-PCR was performed to analyze the Mcl-1 mRNA expression in these cell lines. The RT-PCR results indicated increased expression of Mcl-1 mRNA levels in four human ESCC cell lines compared with that in HaCaT cells ( Figure 1B), which was in agreement with the observations in the immunoblotting analysis. We also performed quantitative real-time RT-PCR to compare mRNA levels of Mcl-1 in these cell lines. As shown in Figure 1C [17] and the interaction of p65 subunit with Naa10p reportedly regulates Mcl-1 expression [18], However, whether NF-κB is involved in Mcl-1 expression in human ESCC cells remains to be clarified. To address this issue, we initially evaluated whether NF-κB is constitutively activated in Mcl-1-expressing human ESCC cells. NF-κB activation as measured by nuclear accumulation has been observed in a wide variety of solid tumors [22]. Therefore, nuclear extracts of TE-1, Eca109, KYSE150, KYSE510 and HaCaT cell lines and the levels of NF-κB subunits in nucleus were estimated. Histone H3 level served as a loading control for nuclear protein [38]. The levels of NF-κB subunits in nuclear extracts of four ESCC cell lines were markedly higher than those in HaCaT cells, suggested that NF-κB is highly constitutively activated in these ESCC cell lines detected. The results indicated that TE-1 cell line displayed relatively high levels of NF-κB subunit p50 and p52. The expression patterns of NF-κB subunit p65, c-Rel and RelB were similar in other three esophageal carcinoma cell lines ( Figure 2). The distinctive patterns for constitutively activated NF-κB subtypes in different ESCC cell lines suggested that NF-κB subunits might play a specific role in regulating Mcl-1 in different esophageal carcinoma cell lines. These results led to the conclusion that the NF-κB pathway is constitutively activated in Mcl-1-expressing human ESCC cell lines.  Mcl-1-κBwt generated higher luciferase activity than that of the pGL2-Basic construct, indicated that high transcriptional activity of human Mcl-1 promoter in three Mcl-1expressing ESCC cell lines tested. However, with a promoter construct mutated at the κB site, the loss of Mcl-1 promoter activity was observed in TE-1 and KYSE150 cells ( Figure 3A). Dominant negative mutants of IκBα (DNMIκBα), a truncant mutant with a deletion of 71 amino acids at the N terminus of IκBα, can competitively inhibit the activation of NF-κB was used to block NF-κB activation as described previously [30]. Expression of DNMIκBα significantly inhibited the Mcl-1 promoter activity in TE-1 and KYSE150 cells ( Figure 3A). Furthermore, compared with their respective DMSO control, treatment with 20 μM Bay11-7082, a specific NF-κB inhibitor, resulted in the Mcl-1 promoter activity drastically curtailed in both TE-1 and KYSE150 cells. The activity of the Mcl-1 promoter with mutated NF-κB site was essentially unaffected by inhibitor treatment ( Figure 3A). NF-κB transcriptional activities in both TE-1 and KYSE150 cell lines have also been estimated by using an NF-κB-driven luciferase reporter. The results indicated that NF-κB-driven luciferase reporter show an increased transcriptional activity in both TE-1 and KYSE150 cells compared with the vector control ( Figure 3B). Bay11-7082 (20 μM) significantly attenuated the increased transcriptional activity of NF-κB-driven luciferase reporter in these two cell lines, thus confirmed the efficiency of Bay11-7082 as an NF-κB inhibitor ( Figure 3B). Notably, the increased transcriptional activity of the Mcl-1 promoter observed in Eca109 cells remained unchanged by the above three strategies ( Figure 3A). Taken together, these results provide consistent evidence that the involvement of NF-κB pathway in the Mcl-1 promoter transcriptional activity in various human ESCC cells.

Binding of transcription factor NF-κB family members to human Mcl-1 promoter
To ascertain whether NF-κB transcription factor can bind the NF-κB site in human Mcl-1 promoter, EMSA was performed with an oligonucleotide probe containing the putative NF-κB binding sequence derived from human Mcl-1 promoter. Three DNA-protein complexes were evident with nuclear extracts from TE-1 cells, labeled bands 1, 2 and 3, respectively ( Figure 5A). To further confirm whether these three bands are specific for the NF-κB complexes, a competition assay was per-  fold molar excess. These results, which were similar to previously published report [39], suggested that the band 3 is specific for the NF-κB complex. The observation that the Mcl-1-κB oligonucleotide can bind non-NF-κB specific complexes as well might due to other protein(s) present in the nuclear extracts that also bind the NF-κB sequence of the oligonucleotide [40]. To identify which components of NF-κB contribute to this binding activity, supershift analysis was performed with nuclear extracts from TE-1 cells. In the presence of antibodies against NF-κB subunits p50, p52, p65, c-Rel, and RelB, the results revealed that the addition of an antibody against p50, p52 or p65 caused a substantial reduction in binding (lanes 7, 8 and 9). The intensity of the DNA-protein complex was slightly depleted by c-Rel (lane 10) while antibody against RelB had no effect on binding (lane 11). IgG control also showed no effect on the intensity of the complex (lane 12). These data demonstrated that binding of these antibodies prevents association with the labeled probe. The decreases in band intensity suggested the presence of these transcription factors in the complex, which indicate that p50, p52 and p65 are the major NF-κB subunits binding to the human Mcl-1-κB probe in vitro.
To determine whether transcription factor NF-κB actually bind to human Mcl-1 promoter in intact cells, we analyzed the fragment that spans the NF-κB binding region within human Mcl-1 promoter using a chromatin immunoprecipitation assay (ChIP). The sheared cross-linked chromatin of TE-1 cells was immunoprecipitated by antibodies specific for NF-κB subunits p50, p52, p65, c-Rel and RelB. An IgG antibody was used as a nonspecific control. The precipitated chromatin DNA was then amplified by PCR using primers specific for NF-κB binding site of human Mcl-1 gene, which produced 200-bp amplicons that could be observed with the positive control (input chromatin) and when the chromatin was precipitated with antibodies for p50 and p65, respectively. No amplification was observed with two negative controls (no chromatin and IgG) ( Figure 5B). The ChIP results indicated that NF-κB subunits p50 and p65 can exert their regulatory function through directly binding to the NF-κB site of human Mcl-1 promoter and finally regulating Mcl-1 expression in TE-1 cells. Overall, the results suggested that the interaction of transcription factor NF-κB subunits p50 and p65 with human Mcl-1 promoter might be a key event in the regulation of Mcl-1 expression in TE-1 cells.

Knockdown of NF-κB subunit attenuates Mcl-1 expression and inhibits TE-1 cell viability
To further confirm the involvement of individual NF-κB subunits in Mcl-1 expression, we performed knockdown experiments. TE-1 cells were transfected with siRNAs to either p50, p65 or a scrambled control and then the Mcl-1 levels were assessed. To determine the optimal time point for analysis, a time-course experiment was performed at multiple time points after transfection. Representative time-course data of Mcl-1 reduced by p50 or p65 siRNA was shown in Figure 6A and B. The levels of endogenous p50 and p65 decreased by 24 h after transfection of si-p50 or si-p65 and peaked 72 h, then gradually recovered with time. The Mcl-1 downregulation peaked 96 h after si-p50 transfection ( Figure 6A) and peaked 72 h after si-p65 transfection ( Figure 6B) and remained at relatively low levels 144 h posttransfection. Base on the timecourse data, the optimal protocol of 72 h-treatment was used in subsequent experiments. Compared with the control siRNA, silencing of p50 or p65 each simultaneously led to a significant decrease of Mcl-1 protein levels ( Figure 6C). With these data confirming the knockdown of NF-κB subunits and the downregulation of Mcl-1 expression, we next tested the effect of the NF-κB subunit siRNAs on TE-1 cell viability. Silencing of p50 or p65 resulted in decrease of Mcl-1 level ( Figure 6D), which significantly inhibited the viability of TE-1 cells ( Figure 6E). Reintroduction of human Mcl-1 ( Figure 6D) significantly restored cell viability ( Figure 6E), indicating that the specific reduction of Mcl-1 by p50 or p65 siRNA. Notably, cell viability was unable to be completely rescued even the Mcl-1 levels were totally recovered, suggesting other NF-κB-dependent proteins might also contribute to TE-1 cell viability. These results suggest that NF-κB subtypes formed functional heterodimers mediating Mcl-1 expression and cell viability in TE-1 cells.  [41]. Another E3 ligase β-TrCP facilitates the ubiquitination and degradation of GSK-3β-phosphorylated Mcl-1, which contributes to GSK-3β-induced apoptosis [42]. Mutational inactivation  of E3 ligase FBW7 was found to occur in several neoplastic diseases, which can decrease Mcl-1 degradation, resulting in increased Mcl-1 protein levels and resistance to chemotherapeutic agents [43]. In contrast, deubiquitinase USP9X, which is overexpressed in some malignancies, stabilizes Mcl-1 and promotes tumor cell survival. Knockdown of USP9X decreased Mcl-1 levels [5]. Moreover, phosphorylation of Mcl-1 at Thr 163 by ERK [43] prolongs the Mcl-1 half-life while phosphorylation at Thr 163 by GSK-3β [42] or Thr 92 by CDK1 [43] enhances Mcl-1 degradation. In addition, Mcl-1 transcripts can be influenced by microRNAs (miRs). For example, miR29b has been demonstrated to downregulate Mcl-1 protein and sensitize cells to apoptosis [44]. Future studies need to explore whether these mechanisms contribute to the elevated Mcl-1 protein in human ESCC. Increased Mcl-1 protein level has been reported to compromise the apoptotic effects of chemotherapeutic agents, resulting in therapeutic resistance [43]. Thus, the pathways that are critical for regulating Mcl-1 expression have been employed to target Mcl-1 for cancer therapy. For instance, in large granular lymphocyte leukemia, targeting Stat3 with its upstream kinase JAKselective inhibitor AG490 transcriptionally suppresses Mcl-1 and promotes apoptosis [12]. PI3K/Akt signaling is involved in Mcl-1 induction [10], targeting this pathway by newly developed PI3K inhibitor PI103 is showed to suppress Mcl-1 and induced apoptosis and restore sensitivity to TRAIL-induced apoptosis in neuroblastoma [45]. Treatment with MEK/ERK inhibitor U0126 resulted in Mcl-1 downregulation and induced marked apoptosis in Mel-RM melanoma cells [46]. Therefore, identification of pathways that regulate Mcl-1 may help to improve the therapeutic effect of chemotherapy. Our data indicated that inhibition of NF-κB pathway by Bay11-7082 ( Figure 4A, B), DNMIκBα ( Figure 4C, D) or NF-κB subunit siRNA ( Figure 6) attenuates Mcl-1 expression in human ESCC cells. We also found that the survival of TE-1 cells is impaired when NF-κB is blocked by expression of p50 siRNA or p65 siRNA and reintroduction of Mcl-1 to the siRNA-transfected TE-1 cells significantly restores cell viability ( Figure 6E). These data that decrease Mcl-1 expression and inhibits cell viability by inhibition of NF-κB pathway support the use of selective NF-κB inhibitors in the treatment of Mcl-1overexpressing human ESCC.

Discussion
By gel shift analysis, nuclear extracts of TE-1 cells were preincubated with antisera directed against individual NF-κB family members p50, p52, p65, c-Rel, RelB or with a nonspecific antisera prior to interaction with the Mcl-1-κB site probe. We found that NF-κB family members p50, p52 and p65 were able to bind to the same probe in vitro. The result was in agreement with the earlier findings that most κB sites show no or little selectivity for a given NF-κB species and different dimers have broad sequence recognition specificities although relatively small differences in the relative affinity of NF-κB dimers for a given site can be found [47][48][49]. However, p50 and p65 but not p52 were revealed directly binding to the κB site of human Mcl-1 promoter in intact cells by ChIP assays. The discrepancy between the measured in vitro affinity of NF-κB for the κB probe and the real in vivo occupancy at κB site of the natural promoter is not without precedent. For instance, ChIP result showed that, in LPS-stimulated DCs, the κB site of IL-8 promoter is a highly selective p65 recruiter [50], while in in vitro experiments, it is bound and activated by both p65 and c-Rel homodimers [51]. The ability of a specific gene to selectively recruit various NF-κB dimers in vivo cannot be predicted on the basis of in vitro results [50]. The context of κB site physiological promoter rather than the κB site itself is the major determinant of which NF-κB dimmer will ultimately be loaded onto a certain promoter.
Although putative binding sites for NF-κB were identified in the Mcl-1 promoter region [9] and two recent reports have shown that NF-κB is directly involved in Mcl-1 regulation [17,18]. In the first article, by using ChIP assay, the authors show that p65 subunit of NF-κB following TRAIL treatment binds to the Mcl-1 promoter, which suggested that TRAIL induced expression of Mcl-1 through activation of NF-κB in HCT-116 colon carcinoma cells [17]. In the second study, the authors show that transcriptional activation of Mcl-1 gene required the recruitment of N-a-Acetyltransferase 10 protein/p65 complex to the p65-binding site of the Mcl-1 promoter region [18]. However, both studies focused only on the role of NF-κB p65 subunit in Mcl-1 expression and the report of other NF-κB subunits involved in Mcl-1 expression is relatively limited. Since dimerization is required for NF-κB binding to DNA and more than 12 homo-and heterodimers have been described [50]. The analysis of other members of the NF-κB family to bind to κB site and regulate Mcl-1 expression would allow for a better understanding of the precise mechanism of Mcl-1 transcriptional control by NF-κB. Our results indicate that effect of NF-κB on Mcl-1 expression in TE-1 cells is due to activation of NF-κB subtypes p65 and p50, without activation of other subtypes ( Figure 5B) and reveal that activations of p65 and p50 are involved in Mcl-1 expression thus affecting cell viability ( Figure 6E). Notably, we did not observe the involvement of NF-κB pathway in human Mcl-1 promoter activity in Eca109 cells ( Figure 3A). In addition to NF-κB binding site, the 325 bp long Mcl-1 promoter fragment contains CRE-BP, Ets, Sp1, SRE, STAT binding sites [17,52]. We speculated that, in Eca109 cells, other transcription factor(s) rather than NF-κB might play a leading role in Mcl-1 expression. Our results suggested that the existence of other regulatory cascades that modulate Mcl-1 expression in different ESCC cells.

Conclusions
In summary, we provided evidence regarding how Mcl-1 is regulated at transcription level in human ESCC cell lines. The present study demonstrated that NF-κB contributes to Mcl-1 production in various human ESCC cells and subunits p50 and p65 of NF-κB positively regulate Mcl-1 expression and cell viability in TE-1 cells. The results support the conclusion that Mcl-1 plays a key role in mediating TE-1 cell fate downstream of the NF-κB pathway. The newly identified mechanism suggests that targeting the NF-κB pathway might improve treatment results in some human ESCCs with high Mcl-1 expression.