Development of an enzyme-linked immunosorbent assay (ELISA) for the specific detection of MEKK1 expression in cellsB

Abstract

Introduction: MEKK1 is a 196-kDa serine-threonine kinase activated in response to a variety of stimuli, including EGF, lysophosphatidic acid, osmotic stress, UV light, and microtubule toxins. However, there are few reports about the expression level of MEKK1 in cancers. Methods: In this report, a direct competitive ELISA to quantify total MEKK1 in cell lines was developed. Results: The procedure showed a high sensitivity (detection limit: 0.17 ng/ml), good precision (coefficient of variation V10.12) and acceptable linearity over a large range of MEKK1 concentrations (0.1–10,000 ng/ml). In a pilot study, this assay was used to quantify MEKK1 in different cell lines. In cancer cells, the range of MEKK1 is 0.02–85 ng/mg protein and its concentration was higher in pancreas cancer cells and umbilical vein cells than that in others. Discussion: This method can be used to measure the MEKK1 level of cell samples. The activity of MEKK1/ JNK pathway in pancreas cancer and umbilical vein cell lines indicates that MEKK1 may be a potential target in interfering with pancreas cancer and angiogenesis.

Keywords: MEKK1; ELISA; Cancer; Angiogenesis

1. Introduction

MEKK1 is a 196-kDa serine-threonine kinase activated in response to a variety of stimuli, including EGF, lysophosphatidic acid, osmotic stress, UV light, and micro- tubule toxins (Fanger, Johnson, & Johnson, 1997; Yujiri et al., 1999). Upon activation, MEKK1 participates in the regulation of the JNK and ERK pathways and is involved in the activation of NF-nB (Lee, Hagler, Chen, & Maniatis, 1997; Meyer, Wang, Chang, Templeton, & Tan, 1996; Yujiri, Sather, Fanger, & Johnson, 1998). In addition, MEKK1 is activated in response to changes in cell shape and the microtubule cytoskeleton (Yujiri et al., 1999). MEKK1 senses microtubule integrity, protects cells from committing to apoptosis, and contributes to the migration of fibroblasts and epithelial cells. Targeted gene disruption of MEKK1 demonstrated that it functionally regulates cell migration both in vivo and in vitro (Yujiri et al., 2000). Consistent with that idea, overexpression of MEKK1 induces the formation of a large lamellipodia-like structure in epithelial cells (Yujiri et al., 2000). However, the mechanism by which MEKK1 influences cell motility remains unclear. Characterization of MEKK1-null mouse embryo fibroblasts demonstrates that MEKK1 regulates the ERK1/2 pathway for control of calpain-catalyzed rear-end detachment (Cuevas et al., 2003).

Enzyme linked immunosorbent assays (ELISAs) in microtiter plate format are widely used for high-throughput screening of chemicals and drugs for their effects on various biological activities. These assays make it possible to rapidly evaluate a large number of compounds and can be readily adapted for automatic manipulations (Kenny, Bushfield, Parry-Smith, Fogarty, & Treherne, 1998; Sundberg, 2000).

In this report, we describe the development of an ELISA to quantify total MEKK1 in human cell lines. The procedure showed a high sensitivity (detection limit: 0.17 ng/ml), good precision (coefficient of variation V10.12) and acceptable linearity over a large range of MEKK1 concentrations (0.1– 10,000 ng/ml). In a pilot study, this assay was used to quantify MEKK1 in different cell lines.

2. Methods
2.1. Expression plasmid

Expression plasmid of human wild-type MEKK1 (pcDNA3-MEKK1) was provided by Dr. Chunhong Yan and subcloned into pcDNA3 (Invitrogen) at EcoRI sites.
The pcDNA3-MEKK1 was digested with EcoRV and EcoRI to get the kinase domain of MEKK1 (deltaMEKK1, amino terminal domain of amino acids 1–297). The bacterial expression vector pET32a (Novagen) was cleaved by the same enzymes. Vector and the fragment were isolated from agarose gel bands using the PURIGENE gel extraction kit (YuanPingHao). Ligation of the fragment in frame with a multifunctional tag (including a hexa-His) in the vector was carried out according to standard procedures using T4 DNA ligase (Biolab) to generate pET32-deltaMEKK1, the thio- redoxin-deltaMEKK1 fusion construct. The ligation mixture was transformed into E. coli DH5a and several clones were screened for plasmid with correct size inserts. Plasmid DNA was isolated from selected colonies and its insert portion was sequenced to ensure correct sequence without any mutations.

2.2. Cell culture

RPMI 1640 media, trypsin and fetal calf serum (FCS) were purchased from Gibco BRL. The cell lines main- tained in RPMI 1640 medium with 2 g/L NaHCO3 supplemented with 10% fetal calf serum, penicillin (100 U/ml), streptomycin (100 Ag/ml) at 37 8C in a humidified air atmosphere containing 5% CO2, were used for in vitro experiments.

2.3. Cell lines

Human umbilical vein cell (ECV304), human proximal tubule epithelial cell (HKC) , Chinese hamster lung cell (CHL) , human embryonic lung cell (HELF), human ovary cancer cell (A2780 and SKOV3), human renal cancinoma cell (KETR3), human gastric carcinoma cell (BGC823), human cervical cancer cell (Hela), human non-small lung cancer cell (A549), human pancreas cancer cell (PC-3), human fibrosarcoma cell (HT1080), human prostate cancer cell (PC3M), human mouth squamous carcinoma cell (KB),vincristin-resistant human mouth squamous carcinoma cell (KBr200, whose IC50 towards VCR is about 200-fold higher than KB’s evaluated by MTT assay), human breast adenocarcinoma cell (MCF-7), adriamycin-resistant human breast adenocarcinoma cell (MCF/Adr), human colon adenoma cell (HT-29), human colonrectal adenocarcinoma cell (HCT-8), human transgenic colonrectal adenocarcinoma cell (m1HCT-8, HCT-8 cells transfected with MEKK1; pHCT-8, HCT-8 cells that transfected with control plasmid pcDNA3.1), mouse melanoma cell (B16), mouse melanoma cell (B16-BL6, derived from B16), mouse melanoma cell transfected with control plasmid pcDNA3.1 by lipofect- AMINE (pB16), mouse transgenic melanoma cell (m1B16, B16 cells transfected with pcDNA3.1-MEKK1). Trans- fections were performed using LipofectAMINE (Life Technologies). Following 2 days, 800 Ag/ml G418 (Life Technologies) was added to the cells, and clones were isolated. Cells were selected in 800 Ag/ml G418 for 4 weeks prior to the start of experiments.
All of the cells above are bought from ATCC or Cell Culture Center of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. The cells were harvested in their exponentially growing phase.

2.4. Expression and purification of the thioredoxin-deltaMEKK1 fusion protein

The pET32-deltaMEKK1 plasmid or pET32a control plasmid was isolated from E. coli DH5a and transformed into E. coli strains BL21 (DE3). The cultures were grown under aerobic conditions at 37 8C in Luria–Bertani (LB) broth with 50 Ag/ml ampicillin until the OD600 reached 0.6–0.8. The expression was then induced with 1 mmol/L isopropyl-h-D-thiogalacto-pyranoside (IPTG). Cells were collected by centrifugation 4 h after induction and resuspended in an ice-cold binding buffer (20 mmol/L sodium phosphate, 0.5 mol/L NaCl, pH 7.8). The resuspended cells were lysed by sonication on ice and the cell debris removed by ultracentrifugation at 30,000 rpm for 30 min.

2.5. Production of the polyclonal antiserum directed against MEKK1

The deltaMEKK1 protein was used for production of rabbit antibodies. The antibodies were produced in two rabbits (R030511, R030512). The rabbits were each immu- nized with 1 mg of deltaMEKK1 mixed with 1 mL PBS four times with 4-week intervals. The titers of the serum from the two rabbits were test and the higher one was used as polyclonal antibody of MEKK1.

2.6. Removal of cross-reactive antibodies

The antiserum of the rabbit for screening was diluted 1:10 with blocking buffer (10 mmol/L Tris–Cl, 150 mmol/L NaCl, 0.05% (v/v) Tween-20, 20% (v/v) fetal bovine serum, pH 8.0). A 0.5 ml of lysate of cells expressing pET32A for every milliliter of antiserum was added and the mixture was
incubated for 4 h at room temperature on a slowly rotating wheel. The treated antibody may be stored at 4 8C in the presence of 0.05% (w/v) sodium azide until use (Sambrook & Russell, 2001).

2.7. Test of the specificity of the antibodies by Western blotting

Cell samples were performed as described in Section

2.11. Aliquots of whole cell lysates were subjected to 10% (or 6% in the case of full-length MEKK1) SDS-PAGE and then transferred to Hybond blotting membranes (Amersham LIFE SCIENCE). The membranes were blocked with 3% bovine serum albumin in Tris-buffered saline containing 0.05% Tween 20 (TTBS) and then probed with the treated antiserum to deltaMEKK1 in 3% bovine serum albumin in TTBS. Positive antibody reactions were visualized using an appropriate alkaline-phosphatase-conjugated secondary antibody and BCIP/NBT luminescence system. The bands were photographed by Kodak transilluminator UV/white 2020D.

2.8. MEKK1 calibrators

The deltaMEKK1 protein was used for preparation of calibrators. Calibrators were prepared from a stock solution of 0.5 g/L deltaMEKK1. This stock solution was diluted in polystyrene test tubes with assay buffer (10 mmol/L Tris, pH 7.6, 150 mmol/L NaCl, 5 mmol/L EDTA pH 8.0, 1% Triton X-100, 0.1 mmol/L PMSF). The assay buffer was used as zero calibrator. Preparing 1:3 serial dilutions of standard deltaMEKK1 antigen solution in blocking buffer. These antigen concentrations will be used in preparing a standard inhibition curve.

2.9. Direct competitive ELISA for quantification of MEKK1

A direct enzyme-linked immunosorbent assay (ELISA) was performed, using deltaMEKK1 as antigen. The ELISA was developed in three steps: (a) the antiserum was tested in a checkerboard design to select the optimal combination of primary and secondary antibodies; (b) the optimal dilution of the detection antibody was established; and (c) the characteristics of the assay were studied.

2.10. Analytical performance of the ELISA procedure

2.10.1. Determination of detection limit, quantitation limit and linearity

The detection limit was defined as the analyte concen- tration corresponding to 90% of the assay response at zero dose. The quantitation limit was defined as the lowest value of the scope of the linearity.

The linearity scope was calculated by unweighted least- squares linear regression analysis of the OD response to the Napierian logarithm of the concentration of the MEKK1 standards, where the coefficient of determination (R2) must be more than 0.980. The software used was Microsoft Excel 2000.

2.10.2. Precision

Within-run (plate=3) and between-day (day=3) preci- sions were assessed using three different dilutions of a purified deltaMEKK1 preparation (deltaMEKK1 concen- trations: 50, 250 and 500 ng/ml) and three different dilutions of cell sample (MEKK1 concentrations: 40, 190 and 380 ng/ml). For each sample, three determinations were performed and the means and standard deviations were calculated. For within-run assay, samples were analyzed in three-well replicates in one day. For between-day assay, samples were analyzed in three-well replicates in three consecutive days.

2.10.3. Analytical recovery

To measure the analytical recovery rate, various amounts of purified deltaMEKK1 (200, 500 and 1000 ng/ml) were added to cell samples containing different concentrations of MEKK1 (38, 193 and 386 ng/ml). MEKK1 concentrations were then measured and the percentage recovery rates were calculated.

2.11. Protein concentrations

Protein concentrations of extracts were determined according to Bradford assay using BSA as standard.

2.12. Preparation of cell samples

All steps of the extraction were performed on ice. Immediately before processing, cells were carefully counted and monitored for viability by trypan blue exclusion method under the microscope, washed three times with ice-cold PBS buffer and harvested. Cells were then lysed by the addition of 1 ml lysis buffer (10 mmol/L Tris, pH 7.6, 150 mmol/L NaCl, 5 mmol/L EDTA pH 8.0, 1% Triton X-100) containing 0.1 mmol/L PMSF. After 30 min on ice, lysates
were collected and clarified by centrifugation at 15,000×g for 10 min at 4 8C.

3. Results
3.1. Expression and purification of deltaMEKK1

In our study, the pcDNA3-MEKK1 kinase domain (deltaMEKK1) was successfully cloned in the pET32a expression vector and transformed to the E. coli BL21(DE3) strain for expression studies. Dose depend- ence and time course studies of the induction of the recombinant protein expression, analyzed by SDS-PAGE, led to an IPTG concentration of 1 mM and induction time of 4 h at 37 8C. High amounts of relatively pure recombinant protein containing the His-tag were recovered and purified from the lysed extract by using immobilized metal affinity chromatography (Fig. 1, lanes 4–6). The reduction of non-specific interactions between extract proteins and the matrix was done by washing the resin with high salt concentration and low imidazole concen- tration buffers. The elution of the recombinant protein was performed using a gradient of imidazole concentration, a procedure that considerably improved the final protein purity. Elution fractions containing the recombinant protein were separated and combined as a pool for further experiments. The purity of the final preparation of deltaMEKK1 was more than 95%, which was estimated by the percent of the relative amount of the band of deltaMEKK1 to the relative total amount of the lane by Gel-Pro Analyzer 3.1 software.

3.2. Characterization of the antibody

As assessed by SDS-PAGE, only the deltaMEKK1 was detected after Coomassie blue staining (Fig. 2, lane 2). The specificity of the antibodies was analyzed by immunoblot- ting using a crude E. coli BL21 (DE3) cell (transformed with pET32-deltaMEKK1 plasmid) extract and purified deltaMEKK1. With testing against a crude cell extract, the polyclonal rabbit antibody labeled no bands other than those corresponding to the deltaMEKK1. It suggests a high specificity of the antiserum (Fig. 2, lane 3). A similar pattern was obtained when the antibody was tested against cancer cell samples (Fig. 3). The 196 kDa full-length MEKK1 could be detected with high specificity.

3.3. Standard curve

A typical standard inhibition curve of direct competitive assay for MEKK1 was shown in Fig. 4. The OD response of it was also listed in Table 1. Concerning the linearity, the concentration scope of deltaMEKK1 chosen in standard curve was from 1.7×10—4 to 1.0×101 Ag/ml, in which scope the coefficient of determination (R2) was more than 0.980. So the quantitation limit of the assay was determined as 0.17 ng/ml, defined as the lowest concentration of the linearity scope. In addition, the detection limit, which was defined as the analyte concentration corresponding to 90% of the assay response at zero dose, was 0.168 ng/ml according to the standard curve.

3.4. Validation of the ELISA procedure

Precision data for within-assay and between-assay was assessed using different dilutions of a cell sample (MEKK1- concentrations: 40, 190 and 380 ng/ml). The coefficient of variation range (expressed as the ratio of the standard deviation/mean values for three replicates) was 5.20–9.54% for within-assay precision (Table 2) and 5.89–10.12% for between-assay precision (Table 3), respectively. When similarly assessed on purified deltaMEKK1 (deltaMEKK1 concentrations: 200, 500 and 1000 ng/ml), the coefficient of variation range was 4.18–6.22 for within-assay precision (Table 2) and 2.81–5.39 for between-assay precision (Table 3). Recovery tests were performed by the addition of purified deltaMEKK1 (200, 500 and 1000 ng/ml) to cell samples containing various amounts of the MEKK1 (38, 193 and 386 ng/ml). The percentage of recovery ranged from 89.23% to 109.32% (Table 4).Assay conditions were the same as those described in Table 1.

3.5. Application of direct competitive ELISA to measure MEKK1 in cell samples

3.5.1. Changes in cell samples that transfected with pcDNA3-MEKK1

The value of the present direct competitive ELISA procedure for quantification of MEKK1 in cells that transfected with pcDNA3-MEKK1 was investigated. The mean MEKK1 level in HCT-8 cells, HCT-8 cells transfected with pcDNA3 control plasmids (pHCT-8) and HCT-8 cells transfected with pcDNA3-MEKK1 plasmids (m1HCT8) was 0.04F0.004 ng/mg (n=3), 0.37F0.03 ng/mg (n=3) and 13.28F1.36 ng/mg (n=3), respectively. The vertical histograms presented in Fig. 5 illustrate the relative MEKK1 level changes in HCT-8, pHCT-8 and m1HCT-8.

The same result was detected in the B16 cells that transfected with pcDNA3-MEKK1. The mean MEKK1 level in B16 cells, B16 cells transfected with pcDNA3 control plasmids (pB16) and in B16 cells transfected with pcDNA3-MEKK1 plasmids (m1B16) was 4.29F0.43 ng/ mg (n=3), 8.69F0.89 ng/mg (n=3) and 88.63F5.99 ng/mg (n=3), respectively. The vertical histograms presented in Fig. 6 illustrate the relative MEKK1 level changes in B16, pB16 and m1B16.

3.5.2. MEKK1 levels in diverse cell lines

MEKK1 levels in cancer cell lines from different tissues were detected. The relative MEKK1 levels in different cell lines were presented in Table 5. The range of MEKK1 is 0.01–85 ng/mg protein. The MEKK1 levels in human pancreas cancer cell (PC-3) and human umbilical vein cell (ECV304) are higher than those in other cell lines.

IgG antibody covalently labeled with AP. Using this method, it was possible to rapidly quantify MEKK1 in cell samples with a nice sensitivity (0.17 ng/ml) and a detection range of approximately 0.1–10,000 ng/ml. Irrespective of the initial concentration of the samples used to test the validity of the present direct competitive ELISA, the assay showed good precision with a coefficient of variation ranging from 2.81 to 10.54 and high recovery between 89.23% and 109.32%. The accuracy was found to be similar for all dilutions of the cell sample analyzed, suggesting that the assay was not affected by the initial protein content of the sample. A good dose–response curve was further obtained between 1.7×10—4 and 1.0×101 Ag/ ml with a coefficient of determination (R2) of 0.9937. Taken together, all data indicates that the assay developed is highly reproducible and sensitive and, hence, may be useful for the routine quantification of MEKK1 in cell samples.

The immunoassay described here was used for the measurement of MEKK1 concentrations in different kinds of cancer cell lines. The MEKK1 concentration was shown to be higher in human pancreas cancer cell (PC-3) and human umbilical vein cell (ECV304) than that in other cell lines. Hirano et al. reported that an analysis of nine pancreatic cancer tissues revealed JNK activation in all tumor samples and ERK activation in three tumor samples. Colony formation assays by transfection of dominant negative mutants of Ras, ERK or MEKK1 into pancreatic cancer cell lines (BxPC-3, PANC-1, MIAPa-Ca-2 and AsPC-1) and an amnion-derived cell line (FL) revealed that DN-MEKK strongly inhibits the survival of colonies in pancreatic cancer cells, but not in FL cells. In vitro kinase assays and luciferase assays using the Gal4 c-Jun system revealed that in pancreatic cancer cells DN— MEKK failed to inhibit JNK activation. In PANC-1 cells, c-Jun was found to be a major component of protein components

4. Discussion

MEKK1 is an important signal transporter in MAPK system. However, there are few reports about the expres- sion level of MEKK1 in cancers. Since no commercial immunochemical kit was available, an ELISA procedure for the accurate quantification of the MEKK1 in cell samples was developed. Quantification of the MEKK1 in cell samples was achieved by using a direct competitive ELISA based on the combination of MEKK1 kinase domain coated on the wells of a microtiter plate together binding to AP-1 site and CRE, but not in FL cells (Hirano et al., 2002).

An aliquot (200 Ag protein/per well) of whole cell lysates was loaded. Assay conditions were the same as those described in Table 1. The data shown are representative of at least three independent experiments.Adenocarcinoma of the pancreas is one of the most aggressive human cancer, it has an extremely poor prognosis. Therefore, a better understanding of the molecular mechanisms leading to this tumor remains a major goal because it may help in devising strategies for earlier diagnosis and better treatment. Over the past decade, several signal pathways have been shown to be important in regulating growth of pancreatic cancer cells. Among these, protein tyrosine kinases, mitogen-activated protein kinases (MAP kinases), the phosphatidylinositide (PI)-3-kinase/protein kinase B (AKT) cascade, and protein kinase C play critical roles in mediating pancreatic cancer cell proliferation, apoptosis, and differentiation(Davie et al., 1999; Krasilnikov, 2000; Nishio et al., 1999; Weinstein et al., 1997). K-ras protooncogene mutation in ras (particularly the Kirsten-ras or K-ras variant) has been described in up to 90% of pancreatic cancer patients (Almoguera et al., 1988).

The different activities of the MEKK1/JNK pathway in pancreatic cancer cells and non-pancreatic cancer cells suggested that the MEKK1 pathway mainly contributes to cell survival in pancreatic cancer cells and may provide an advantage for the therapy of pancreatic cancers treated with drugs inhibiting this pathway.

The formation of new blood vessels out of pre-existing capillaries, or angiogenesis, is a sequence of events that is of key importance in a broad array of physiologic and pathologic processes. A large number of different and nonrelated diseases is associated with formation of new vasculature. Among these pathologies are diseases, such as tissue damage after reperfusion of ischemic tissue or cardiac failure, where angiogenesis is low and should be enhanced to improve disease conditions(Carmeliet et al., 1999; Ferrara & Alitalo, 1999). In several diseases, excessive angio- genesis is part of the pathology. These diseases include cancer (both solid and hematologic tumors), cardiovascular diseases (atherosclerosis), chronic inflammation (rheuma- toid arthritis, Crohn’s disease), diabetes (diabetic retinop- athy), psoriasis, endometriosis, and adiposity. These diseases may benefit from therapeutic inhibition of angio- genesis (Folkman, 1995; Hanahan & Folkman, 1996). The high level of MEKK1 in ECV304 shows that MEKK1 may be a suitable target in angiogenesis.

In conclusion, an ELISA to quantify total MEKK1 in human cell lines was developed. The procedure showed a high sensitivity (detection limit: 0.17 ng/ml), good precision (coefficient of variation V10.12) and acceptable linearity over a large range of MEKK1 concentrations (0.1–10,000 ng/ml). In a pilot study, this assay was used to quantify MEKK1 in different human cell lines. In cancer cells, the range of MEKK1 is 0.01–85 ng/mg protein and its concentration was higher in pancreas cancer and umbilical vein cells than that in others. In addition, the activity of MEKK1/JNK pathway in these two cell lines indicates that MEKK1 A-196 may be a potential target in interfering with pancreas cancer and angiogenesis.