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ISSN 0582-9879                                    ACTA BIOCHIMICA et BIOPHYSICA SINICA 2003, 35(1): 6-12                                    CN 31-1300/Q

　

Preparation of an Anti-Cdx-2 Antibody for Analysis of Different Species Cdx-2 Binding to acat2 Promoter

SONG Bao-Liang1, QI Wei1,2, WANG Can-Hua1,3, YANG Jin-Bo1, YANG Xin-Ying1, LIN Zhi-Xin3, LI Bo-Liang1*

( 1 State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology,

Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200031, China;

2 Department of Biological Science and Technology, Nanjing University, Nanjing 210093, China;

3 Department of Biological Science and Technology, Shanghai Jiaotong University, Shanghai 200030, China )

 

Abstract      The homeodomain protein, Cdx-2, as transcription factor has been implicated in the transcriptional regulation of genes expressed in small intestine and the process of tumorgenesis. In current work, a conserved mouse Cdx-2 domain (mCdx-2D) coded by its cDNA fragment, which was amplified and cloned into the expression vector pGEX-4T-1, was expressed as a fusion protein with GST (GST-mCdx-2D) and purified by one step of affinity chromatography. A polyclonal antibody against Cdx-2 was raised by using the recombinant fusion protein GST-mCdx-2D as antigen and was fractionated from the rabbit anti-serum. Western blot and EMSA (electrophoretic mobility shift assay) demonstrate that the natural and denatured Cdx-2s from different species (mouse and human) can be detected by the prepared anti-Cdx-2 antibody. Most notably, we found that the Cdx-2 in human intestine cell line Caco-2 is expressed in a differentiation-dependent manner and can efficiently bind to the mouse and human acat2(acyl-coenzyme A: cholesterol acyltransferase 2) promoter regions, suggesting that the transcriptional factor Cdx-2 may play a role in regulating the acat2 expression in the intestinal cells.

 

Key words     Cdx-2; GST fusion protein; polyclonal antibody; EMSA; acat2 promoter

 

The caudal-related homeodomain protein, Cdx-2, is crucial in intestinal cell differentiation and tumorgenesis[1–5]. It is also the first identified transcription factor[6], most likely acting in conjunction with a network of other factors[7-9], which are responsible for regulating expressions of many intestine-specific genes including sucrase-isomaltase(SI)[6] and others[10-16]. Cdx-2 is expressed in a complex pattern in developing mouse embryo[17] and at high level in the intestinal epithelium of adult mice[17, 18]. However, the distribution of Cdx-2 in the differentiating Caco-2 cell, which is a colon cacinoma cell line that can differentiated into enterocyte cell when getting confluent[19], has not been determined so far.

On the other hand, ACAT (acyl-CoA: cholesterol acyltransferase) is an intracellular enzyme responsible for catalyzing the intracellular formation of cholesteryl esters from cholesterol and long-chain fatty acyl-coenzyme A[20]. This enzyme participates in various physiological processes such as lipoprotein assembly and dietary cholesterol absorption[21,22], and plays a very important role in development of foam cell as the early stage of atherosclerosis[23] and in modulation of the amyloid-beta polypeptide generation (Aβ) in Alzheimer’s disease[24]. In mammals, two ACAT genes have been identified[25–27]. In adult, ACAT1 protein is found almost in all of the examined cells and tissues, including hepatocytes and Kupffer cells of liver, adrenal gland, skin, intestines, and macrophages. In contrast, ACAT2 protein is selectively expressed in liver and intestine. It is mainly found in the apical region of the intestinal villi and in the differentiated Caco-2 cells[28,29]. Previous study showed that the high activity of human acat2 promoter in the differentiated Caco-2 cells might be related to Cdx-2 elements[30].

In this paper, we described the preparation of a specific anti-Cdx-2 antibody that can be used to detect different species of Cdx-2 proteins and the binding of the Cdx-2 to various DNA probes including human and mouse acat2 promoter regions.

1    Materials and Methods

1.1  Materials

1.1.1       Plasmids, bacteria and cell lines   The E. coli GST expression vector pGEX-4X-1 was the product of Amersham Pharmacia Biotech Company. The plasmid pRc/Cdx-2 containing the mouse Cdx-2 coding region was a generous gift from Dr. PG Traber of the University of Pennsylvania (USA). The plasmid pRc/CMV was obtained from Invitrogen (USA). The E.coli strain BL21(DE3) was stored in our laboratory. Following cell lines were used in this study: Caco-2 cell (a human colon carcinoma cell line that spontaneously differentiates into an absorptive enterocyte phenotype after reaching confluence)[19], HeLa cell (a cell line derived from cervical cancer)[31], hepatoblastoma cell line HepG2[32,33], and CHO cell line AC29[34]. All the cells were grown as described previously[30–35].

1.1.2       Enzymes and other reagents   All the tissue culture media, trypsin, tissue culture dishes, fetal bovine serum (FBS) and T4 DNA ligase were purchased from Gibco BRL. All the restriction enzymes and agarose were from Premega. Taq DNA polymerase and dNTPs were from Sino-American Biotech (Shanghai, China). GSH-Sepharose-4B was from Pharmacia. Isopropylthio-β-D-galactoside (IPTG) and other reagents were ordered from Sigma. All the oligonucleotides were synthesized with an automated DNA synthesizer in Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences.

1.2   Methods

1.2.1       Construction of GST-fusion expression plasmid pGEX-mCdx-2D       The plasmid pRc/Cdx-2 containing the whole coding region of mouse Cdx-2 gene was from Dr. PG Traber[6]. We amplified the mouse Cdx-2 cDNA fragment from the plasmid pRc/Cdx-2 by PCR with the forward primer 1 (5'-GTG-GGATCCGCGGCGGCTGCTA-3', in which the BamHI site was underlined) and reverse primer 2 (5'-GCGG-CTCGAGATGCGGGTGATGGTG-3', in which the XhoI site was underlined). The amplification was performed by using a procedure of 30 cycles of reaction with denaturing at 94 ºC for 30 s, annealing at 55 ºC for 30 s and extension at 72 ºC for 1 min. After digested with the restriction endonucleases BamHI and XhoI, the partial Cdx-2 cDNA (216 bp) and the GST-fusion expression vector pGEX-4T-1 were linked by T4 DNA ligase. The positive GST-mCdx-2D fusion expression plasmid was identified by restriction endonuclease digestion and further verified by DNA sequencing on an automatic DNA sequencer by Genecore Biotech (Shanghai, China).

1.2.2       Expression and solubility analysis of GST-mCdx-2D fusion protein   A 37 ºC overnight starter culture of E. coli BL21 (DE3) transformant harboring the expression plasmid was inoculated into Luria-Bertani medium containing 50 mg/L ampicillin to a final concentration of 2% and allowed to grow at 37 ºC under vigorous shaking (250 r/min). After the A600 of the culture reached above 0.5, IPTG was added to a final concentration of 0.5 mmol/L, and the culture was moved to 22 ºC and allowed to grow for another 8 hours. The cells were harvested by centrifugation, washed with ice-cold phosphate-buffered saline (PBS) and lysed by a sonicator equipped with an appropriate probe. The lysate was centrifuged for 18 min at 18 000 r/min (4 ºC). The supernatant and pellet were used in SDS-PAGE analysis to determine the solubility of the expressed protein.

1.2.3       Purification of GST-mCdx-2D fusion protein by affinity chromatography        This part of the work was performed by using GSH-Sepharose-4B and following the manual provided by Pharmacia Company.

1.2.4       Immunogenecity with GST-mCdx-2D fusion protein as antigen   The purified fusion protein was separated by SDS-PAGE gel, and then the gel was stained with 250 mmol/L KCl solution for 10 min[36]. The protein band with 34 kD size was cut and used in immunogenesis. New Zealand white rabbit was immunized with the purified GST-mCdx-2D protein. 200 μg of protein was emulsified in Freund complete adjuvant and was administered by intradermal injection. Rabbit was given booster injections of 50-100 μg of protein with Freund incomplete adjuvant every other week. The rabbit was bled prior to immunization (pre-immune serum) and after the last immunization (immune serum). IgG was fractionated from the rabbit anti-serum by precipitation with 40% saturated ammonium sulfate and stored at 4 ºC [37].

1.2.5       Removal of the anti-GST antibody from IgG solution  After desalinization, IgG solution was freed of anti-GST antibody by affinity gel filtration on GST-binding agarose (AG-GST). The affinity column was prepared by binding of GST to GSH-Sepharose-4B in PBS, pH 7.2. IgG solution was applied to a column of AG-GST at 4 ºC for 30 min. Then, the flow-through parts were collected and screened by ELISA.

1.2.6ELISAThe rabbit anti-serum and antibody were tested against antigen adsorbed to polystyrene U-well by ELISA. The experiments were conducted as described in reference [38]. Pre-immune serum was included as negative control.

1.2.7       Transient expression of mouse Cdx-2 in CHO cells      The eukaryotic expression plasmid pRc/Cdx-2 that contains the coding region of Cdx-2 gene directed by CMV promoter and control vector pRc/CMV were transfected into CHO cells by using the calcium phosphate co-precipitation protocol[30,35]. Briefly, the cells were seeded in 60-mm tissue culture plates at the density of 5×105 cells/plate the day before transfection. One hour before transfection, fresh medium was added. The DNA/calcium phosphate precipitates containing 9 μg of expression plasmid were added dropwise to the medium and incubated with the cells at 37 ºC for 11 h. Then, the cells were rinsed twice with PBS, and were further grown for 48 hours in the culture medium.

1.2.8       Preparation of nuclear extracts and analysis of Western blot      Preparation of nuclear extracts was performed according to the method developed by Andrews et al.[39] with slight modifications.

Protein samples were boiled for 5 min in Laemmli buffer containing 20 g/L SDS and 100 mmol/L DTT, separated by SDS-PAGE and transferred to nitrocellulose filters by semi-dry electroblotting. The transferred nitrocellulose filters were saturated for 1 hour at room temperature in TBS containing 50 g/L nonfat milk and 0.5 g/L Tween 20. For immunological detection, the treated filters were incubated for 4 h at room temperature with purified anti-Cdx-2 antibody in TBS containing 10 g/L nonfat milk and 0.5 g/L Tween 20, and subsequently with secondary anti-rabbit antibody labeled by HRP (Santacruz Co.). Detection by chemiluminescence was performed by using Western blotting detection reagents (Amersham).

1.2.9       Electrophoretic mobility shift assay (EMSA)        For EMSA with recombinant Cdx-2, complementary oligonucleotides with overhung ends were synthesized, annealed, and labeled by filling in the ends with [32P]-labeled dNTPs and Klenow enzyme. The oligonucleotides used were synthesized according to the sequence of SIF1 element in SI gene promoter that had two Cdx-2 binding sites. The sequences of oligonucleotides were shown (Top strand: 5'-GATCCGTGCAATAAAACTTTATGAGTAA-3'/ Bottom strand: 5'-GCACGTTATTTTGAAATACTC-ATTCTAG-3').

For study on the binding of Cdx-2 to acat2 promoter regions, the DNA fragment with Cdx-2 elements of human and mouse acat2 promoter were labeled with [α-32P]-dATP by the 3'-end filling reaction.

Nuclear extracts were incubated for 30 min at 25 ºC in 10 μl of binding buffer (10 mmol/L Tris-HCl, pH 7.5, 1 mmol/L MgCl2, 40 g/L glycerol, 50 mmol/L NaCl, 0.5 mmol/L DTT, 0.5 mmol/L EDTA, 3 μg of poly(dI)-(dC) from Amersham Pharmacia Biotech Inc.). 1 ng of labeled probe (≈1´104 dpm) was added to the binding reaction mixture and incubated at 25 ºC for 20 min. The bound mixtures were size-fractionated on a non-denatured, 4.5% polyacrylamide gel (29:1, mas:mass, acrylamide: N, N'-methylenebisacrylamide), running at 200 V for 3 h in 0.5´TBE buffer. The gel was subsequently dried and autoradiographed with phosphor-image scanning system. For competition experiments, the binding reaction was incubated with excessive unlabelled probe for 10 min and then the labeled probe was added. For ‘supershift’ analysis, 1 μl of antibody was added to the binding reaction 30 min before addition of probe[35].

 

Fig.1       Preparation of the fusion protein GST-mCdx-2D

（A）Homologous comparison between amino acid sequences of human (h) and mouse (m) Cdx-2 fragments. Numbers representing the amino acid positions of Cdx-2 protein were shown. Different amino acids were underlined. （B）Diagram showing the mouse Cdx-2 and GST-mCdx-2D fusion protein. （C）SDS-PAGE analysis of the expressed and purified GST-mCdx-2D fusion protein. 1, bacterial sample before induction by IPTG; 2, bacterial sample after induction by IPTG; 3, molecular weight markers; 4, supernatant of lysed bacteria; 5, pellet of lysed bacteria; 6, flow-through of supernatant after affinity chromatography by Sepharose-4B glutathine; 7, purified GST-mCdx-2D from soluble proteins.

 

2    Results

2.1  Expression of GST-mCdx-2D fusion protein in E. coli

The mouse Cdx-2 cDNA fragment coding the conserved domain of Ala48 to His119 (Fig.1) with only 5 different amino acids (underlined) was amplified by PCR and inserted into the E. coli GST fusion expression vector pGEX-4T-1 (Amersham Pharmacia Biotech). The positive GST-mCdx-2D fusion expression plasmid, termed as pGEX-mCdx-2D, was identified by restriction endonuclease digestion and further verified by DNA sequencing (data not shown). The recombinant GST-mCdx-2D fusion protein and the amino acid sequences of conserved mouse and human Cdx-2Ds are shown in Fig.1(A) and 1(B). The E.coli BL21 (DE3) host cells harboring the GST fusion expression plasmid pGEX-mCdx-2D were induced by IPTG at 22 ºC for 8 hours to get more soluble amounts of GST-mCdx-2D fusion proteins [Fig.1(C)]. By one step of affinity chromatography with GSH-Sepharose-4B column, the purified GST-mCdx-2D protein [Fig.1(C)] was obtained as about 15 mg per liter bacterial culture.

 

Fig.2       Immunological reactivity of the antisera and the antibody

(A) Immunological reactivity of pre-immune serum (-) and immune serum (…) with GST-mCdx-2D (●) and GST (○). (B) ELISA with the GST-mCdx-2D (●) or GST (○) as antigen and the fractionated IgG (-) or anti-GST antibody removed IgG (…) as antibody.

 

2.2  Preparation of the anti-Cdx-2 antibody

Two New Zealand rabbits were immunized with the purified GST-mCdx-2D. The antibodies in the rabbit sera were detected by ELISA. The pre-immune serum had no immunological reaction with GST-mCdx-2D or GST, but the avidity of the immune serum towards GST-mCdx-2D and GST were 1:5´104 and 1:2´104, respectively[40] [Fig.2(A)]. To improve the specificity and the efficacy of the antiserum, we first prepared the crude IgG from antiserum with ammonium sulfate precipitation, then the GST antibody were removed. As shown in Fig.2(B), the purified antibody had nearly no immunological reaction with GST but reacted strongly with GST-mCdx-2D fusion protein.

 

Fig.3 Binding of the prepared anti-Cdx-2 antibody to different Cdx-2s in Western blot

(A) Recombinant mouse Cdx-2 analysis. 1, nuclear proteins from CHO cells transfected with pRC/CMV plasmid as negative control; 2-4, total, nuclear and cytosol proteins of CHO cells transfected with the mouse Cdx-2 expression plasmid. (B) Endogenous human Cdx-2 analysis. The cellular proteins were prepared from the Caco-2 cells differentiated for 4 days. 1, total proteins; 2, cytosol proteins; 3, nuclear proteins; 4, negative control. (C) Examination of human Cdx-2 in various cell lines. Nuclear extracts were prepared from the undifferentiated (lane 1), 2-day-differentiated (lane 2), 4-day-differentiated (lane 3) Caco-2 and HepG2 cells (lane 4) and HeLa cells (lane 5).

 

2.3  Immunodetection of the denaturalized Cdx-2s by the prepared antibody

The recombinant mouse Cdx-2 expressed in mammalian cells and endogenous human Cdx-2 in Caco-2 cells were analyzed by Western blot analysis with the prepared antibody. As shown in Fig.3(A) and (B), mouse and human Cdx-2 existed in both the whole cell lysate and the nuclear protein extracts, but were not detected in the cytosol proteins, indicating that the Cdx-2 was mainly in the nucleus.

The antibody was then used to analyze the endogenous expression of Cdx-2 in a variety of human cell lines including HeLa, HepG2 and Caco-2 (a human colon carcinoma cell line that can spontaneously differentiate into the entorocytes after getting confluent[19]). In HeLa and undifferentiated Caco-2 cells, there were moderate expressions of Cdx-2 [Fig.3(B), lane 1 and 5]; the highest expressions of Cdx-2 were observed in differentiated Caco-2 cells [Fig.3(B), lane 2 and 3]; but no Cdx-2 was detected in HepG2 cells [Fig.3(B), lane 4]. As far as we know, it is the first time to analyze the expression of Cdx-2 in these cells by Western blot analysis. According to our data, the expression of Cdx-2 is of cell-type specificity and differentiation-dependence in Caco-2 cells. And Cdx-2 is preferentially expressed in the differentiated Caco-2 cells.

2.4  Binding of the natural Cdx-2 to DNA probes analyzed by the prepared antibody

To identify whether the antibody can recognize the natural Cdx-2 protein, EMSA was performed with the nuclear extracts from the CHO cells transfected with the plasmid pRC/Cdx-2. In this experiment, synthesized SIF-1 element containing two Cdx-2 binding sites (TTTAT) was used as probe that was located in the promoter region of human sucrase-isomaltase gene which is regulated by Cdx-2 in small intestine[4, 8]. As shown in Fig.4(A), two shift bands were observed (lane 2) for either one or two Cdx-2 molecular banded to the probe. When the antibody was applied, supershift bands can be observed clearly; meanwhile the specific binding bands were diminished [Fig.4(A), lane 4]. The results evidently demonstrate that the antibody can recognize the natural Cdx-2 and will be very useful in the functional research of Cdx-2.

Similar to Cdx-2, ACAT2 protein was highly expressed in the intestinal cells[28]. Sequence analysis revealed there were three Cdx-2 elements in the mouse acat2 promoter fragment (–869 to –569 bp) and four Cdx-2 elements in the human acat2 promoter region (–912 to –768 bp). To examine the functional significance of the Cdx-2 elements in acat2 promoter, we isolated nuclear extracts from Caco-2 cells differentiated for 4 days, and performed EMSA using the mouse acat2 promoter fragment (–869 to –569 bp) or human acat2 promoter fragment (–912 to –768 bp) as labeled probes. The results [Fig.4 (B) and(C)] illustrated that two specific bands were detectable (lane 2); the binding was eliminated upon incubation with excess unlabeled probe (lane 3); and was supershifted by incubation with the anti-Cdx-2 antibody (lane 4). These results strongly indicated that mouse and human acat2 might be transcriptionally regulated by Cdx-2 in intestinal cells.

 

Fig.4       Supershift binding of the prepared anti-Cdx-2 antibody to different Cdx-2 in EMSA

The synthesized SIF-1 probe (A), mouse (B) and human (C) acat2 promoter regions were labeled and 1×104 dpm of labeled probes were used for each binding reaction. 1, [32P]-labeled probe alone; 2, binding reaction between the labeled probes and Cdx-2 in nuclear extracts prepared from the 4-day-differentiated Caco-2 cells; 3, competition by adding 100-fold molar excess of cold probe to the binding reaction described in lane 2; 4, supershift reaction by adding 1 μl of the prepared anti-Cdx-2 antibody to the binding reaction described in lane 2. Supershift bands were indicated. (A) Binding of the synthesized SIF-1 DNA fragment containing consensus Cdx-2 elements to recombinant mouse Cdx-2 from CHO cells transfected with mouse Cdx-2 expression plasmid. (B) Binding of the mouse acat2 promoter region containing Cdx-2 elements [indicated by three short black bars in Fig. 4(B)] to human Cdx-2 from Caco-2 cells. (C) Human Cdx-2 from Caco-2 cell nuclear extract bound to the human acat2 promoter region containing Cdx-2 elements [indicated by four short black bars in Fig.4(C)]. Supershift bands were indicated by arrows.

 

3    Discussion

Cdx-2 is an intestinal-specific transcription factor that plays an important role in proliferation and differentiation of intestinal cells, as well as in tumorgenesis. Cdx-2 contains a highly conserved homeodomain (aa 218-241) among many homeoproteins, which might decrease the specificity of the resulting antibody. Therefore, we chose the fragment (Ala48-His119) instead of the full length of mouse Cdx-2 as antigen to raise the antibody. In addition, both human and mouse Cdx-2s shared a high degree of homology in this region (67 are same among 72 aa) [Fig.1(A)]. So, the resulting antibody will not react with other homeoproteins and can be used to analyze human Cdx-2 specifically though we utilized the mice’s as antigen.

Cdx-2 is a kind of transcriptional regulator that is localized in nucleus, which has been confirmed by our experimental results [Fig.3(A) and (B)]. The conserved amino-terminal sequences Asp-Arg-Asp [Fig.1(B)] have been identified as a nuclear export signal that mediates Cdx-2 compartmentalization[41]. Furthermore, the endogenous expression of Cdx-2 in various human cell lines was examined. To our knowledge, it is the first time to detect its expression in these cell lines by Western blot analysis. Our results are consistent with those obtained by Southwestern blot[42], Northern blot, in situ hybridization and immunohistochemical detection[18] in mouse. Notably, the expression of Cdx-2 increases greatly during the differentiation of Caco-2 cells.

In Caco-2 cells, the expressional pattern of Cdx-2 is quite similar to human ACAT2[28] which participates in the cholesterol absorption and the assembly of apoB-containing lipoprotein. Previous study showed that ACAT2 was preferentially expressed in the intestine and the differentiated Caco-2 cells[28]. We found its promoter activity is extremely high in differentiated Caco-2 cells[30]. Sequence analysis by computer showed that three Cdx-2 elements were located in mouse acat2 promoter region and four in human acat2 promoter. EMSA demonstrated that both mouse and human acat2 promoter fragment were able to form two specific DNA-protein complex in which Cdx-2 was involved [Fig.4(B) and (C)]. However, we could not illustrate which Cdx-2 element(s) was functionally important for the regulation of acat2 promoter at present. It is further required to test the relative importance of each Cdx-2 element in acat2 promoter.

In this paper, we prepared the specific anti-Cdx-2 antibodies against a conserved domain of mouse Cdx-2. Several methods including Western blot and EMSA were used to identify the antibody. We also analyzed the expression of Cdx-2 in various cell lines including Caco-2 and demonstrated the binding of Cdx-2 to mouse and human acat2 promoters.

 

Acknowledgements     We thank Prof. TRABER Peter G. (University of Pennsylvania, USA) for the generous gift of pRC/Cdx-2 plasmid, Prof. ZHANG Zu-Chuan for the Freund adjuvant, FANG Chang-Ming and YANG Qi-Heng for the helpful discussion. We also thank our colleagues YANG Li, MA Han-Hui, YAO Wei, ZHANG Nian-Yi, JIANG Ying, YAO Xiao-Min and ZHANG Wen-Jing.

 

References

 

1     Beck F, Erler T, Russell A, James R. Expression of Cdx-2 in the mouse embryo and placenta: Possible role in patterning of the extra-embryonic membranes. Dev Dyn, 1995, 204(3): 219-272

2     Suh E, Traber PG. An intestine-specific homeobox gene regulates proliferation and differentiation. Mol Cell Bio, 1996, 16(2): 619-625

3     Chawengsaksophak K, James R, Hammond VE, Kontgen F, Beck F. Homeosis and intestinal tumours in Cdx2 mutant mice. Nature, 1997, 386(6620): 84-87

4     Mallo GV, Rechreche H, Frigerio JM, Rocha D, Zweibaum A, Lacasa M, Jordan BR et al. Molecular cloning, sequencing and expression of the mRNA encoding human Cdx1 and Cdx2 homeobox. Down-regulation of Cdx1 and Cdx2 mRNA expression during colorectal carcinogenesis. Int J Cancer, 1997, 74(1): 35-44

5     Lorentz O, Cadoret A, Duluc I, Capeau J, Gespach C, Cherqui G, Freund JN. Downregulation of the colon tumour-suppressor homeobox gene Cdx-2 by oncogenic ras. Oncogene, 1999, 18(1): 87-92

6     Suh E, Chen L, Taylor J, Traber PG. A homeodomain protein related to caudal regulates intestine-specific gene transcription. Mol Cell Biol, 1994, 14(11): 7340-7351

7     Hussain MA, Habener JF. Glucagon gene transcription activation mediated by synergistic interactions of pax-6 and Cdx-2 with the p300 co-activator. J Biol Chem, 1999, 274(41): 28950-28957

8     Andersen FG, Heller RS, Petersen HV, Jensen J, Madsen OD, Serup P. Pax6 and Cdx2/3 form a functional complex on the rat glucagon gene promoter G1-element. FEBS Lett, 1999, 445(2-3): 306-310

9     Ritz-Laser B, Estreicher A, Klages N, Saule S, Philippe J. Pax-6 and Cdx-2/3 interact to activate glucagon gene expression on the G1 control element. J Biol Chem, 1999, 274(7): 4124-4132

10    Jin T, Drucker DJ. Activation of proglucagon gene transcription through a novel promoter element by the caudal-related homeodomain protein Cdx-2/3. Mol Cell Biol, 1996, 16(1): 19-28

11    Jin T, Trinh DK, Wang F, Drucker DJ. The caudal homeobox protein Cdx-2/3 activates endogenous proglucagon gene expression in InR1-G9 islet cells. Mol Endocrinol, 1997, 11(2): 203-209

12    Lambert M, Colnot S, Suh E, L’Horset F, Blin C, Calliot ME, Raymondjean M et al. Cis-acting elements and transcription factors involved in the intestinal specific expression of the rat calbindin-D9K gene: Binding of the intestine-specific transcription factor Cdx-2 to the TATA box. Eur J Biochem, 1996, 236(3): 778-788

13    Colnot S, Romagnolo B, Lambert M, Cluzeaud F, Porteu A, Vandewalle A, Thomasset M et al. Intestinal expression of the calbindin-D9K gene in transgenic mice. Requirement for a Cdx2-binding site in a distal activator region. J Biol Chem, 1998, 273(48): 31939-31946

14    Barley NF, Prathalingam SR, Zhi P, Legon S, Howard A, Walters J R. Factors involved in the duodenal expression of the human calbindin-D9k gene. Biochem J, 1999, 341 (Pt 3): 491-500

15    Drummond F, Sowden J, Morrison K, Edwards YH. The caudal-type homeobox protein Cdx-2 binds to the colon promoter of the carbonic anhydrase 1 gene. Eur J Biochem, 1996, 236(2): 670-681

16    Drummond FJ, Sowden J, Morrison K, Edwards YH. Colon carbonic anhydrase 1: Transactivation of gene expression by the homeodomain protein Cdx2. FEBS Lett, 1998, 423(2): 218-222

17    James R, Erler T, Kazenwadel J. Structure of the murine homeobox gene Cdx-2. Expression in embryonic and adult intestinal epithelium. J Biol Chem, 1994, 269(21): 15229-15237

18    James R, Kazenwadel J. Homeobox gene expression in the intestinal epithelium of adult mice. J Biol Chem, 1991, 266(5): 3246-3251

19    Levy E, Mehran M, Seidman E. Caco-2 cells as a model for intestinal lipoprotein synthesis and secretion. FASEB J, 1995, 9(8): 626-635

20    Chang TY, Chang CC , Cheng D. Acyl-coenzyme A: Cholesterol acyltransferase. Annu Rev Biochem, 1997, 66: 613-638

21    Suckling KE, Stange EF. Role of acyl-CoA: Cholesterol acyltransferase in cellular cholesterol metabolism. J Lipid Res, 1985, 26: 647-671

22    Dietschy JM, Turley SD, Spady DK. Role of liver in the maintenance of cholesterol and low density lipoprotein homeostasis in different animal species, including humans. J Lipid Res, 1993, 34: 1637-1659

23    Tabas I. The stimulation of the cholesterol esterification pathway by atherogenic lipoproteins in macrophages. Curr Opin Lipidol, 1995, 6: 260-268

24Puglielli L, Konopka G, Pack-Chung E, Chang TY, Tanzi RE, Kovacs DM. Acyl-coenzyme A: Cholesterol acyltransferase modulates the generation of the amyloid β-peptide. Nat Cell Biol, 2001, 3: 905-912

25    Anderson RA, Joyce C, Davis M, Reagan JW, Clark M, Sheiness GS, Rudel LL. Identification of a form of acyl-CoA: Cholesterol acyltransferase specific to liver and intestine in nonhuman primates. J Biol Chem, 1998, 273: 26747-26754

26    Cases S, Novak S, Zheng YW, Myers HM, Lear SR, Sande E, Welch CB et al. ACAT-2, a second mammalian acyl CoA: Chelesterol acyltransferase: Its cloning, expression, and characterization. J Biol Chem, 1998, 273: 26755-26764

27    Oelkers P, Behari A, Cromley D, Billheimer JT, Sturley SL. Characterization of two human genes encoding acyl coenzyme A: Cholesterol acyltransferase-related enzymes. J Biol Chem, 1998, 273: 26765-26771

28    Chang CC , Sakashita N, Ornvold K, Lee O, Chang ET, Dong R, Lin S et al. Immunological quantitation and localization of ACAT-1 and ACAT-2 in human liver and small intestine. J Biol Chem, 2000, 275: 28083-28092

29    Lee O, Chang CC, Lee W, Chang TY. Immunodepletion experiments suggest that acyl-coenzyme A: Cholesterol acyltransferase-1 (ACAT-1) protein plays a major catalytic role in adult human liver, adrenal gland, macrophages, and kidney, but not in intestines. J Lipid Res, 1998, 39: 1722-1727

30    Song BL, Qi W, Yang XY, Chang CC, Zhu JQ, Chang TY, Li BL. Organization of human ACAT-2 gene and its cell-type-specific promoter activity. Biochem Biophys Res Commun, 2001, 282(2): 580-588

31    Chen TR. Re-evaluation of HeLa, HeLa S3, and HEp-2 karyotypes. Cytogenet Cell Genet, 1988, 48(1): 19-24

32    Knowles BB, Howe CC, Aden DP. Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science, 1980, 209(4455): 497-499

33    Busch SJ, Barnhart RL, Martin GA, Flanagan MA, Jackson RL. Differential regulation of hepatic triglyceride lipase and 3-hydroxy-3- methylglutaryl-CoA reductase gene expression in a human hepatoma cell line, HepG2. J Biol Chem, 1990, 265(36): 22474-22479

34    Cadigan KM, Heider JG, Chang TY. Isolation and characterization of Chinese hamster ovary cell mutants deficient in acyl-coenzyme A: Cholesterol acyltransferase activity. J Biol Chem, 1988, 263(1): 274-282

35    Yang JB, Duan ZJ, Yao W, Lee O, Yang L, Yang XY, Sun X et al. Synergistic transcriptional activation of human ACAT-1 gene by interferon-g and all-transretinoic acid in THP-1 cells. J Biol Chem, 2001, 276(24): 20989-20998

36    Adams LD, Weaver KM. Detection and recovery of proteins from gels following zinc chloride staining. Appl Theor Electrophor, 1990, 1(5): 279-282

37    Green JA, Manson MM. Production of polyclonal antisera. Methods Mol Biol, 1998, 80: 1-4

38    Hancock DC, Evan GI. Production and characterization of antibodies against synthetic peptides. Methods Mol Biol, 1998, 80: 15-22

39    Andrews CN, Faller DV. A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucl Acids Res, 1991, 19(9): 2499

40    Xie F, Ping BF, Zhang ZC, Zhang YL. Expression of fusion protein containing androgen receptor segment and preparation of polyclonal antibodies to androgen receptor. Acta Biochim Biophys Sin, 1998, 30(4): 382-386

41    Trinh KY, Jin T, Drucker DJ. Identification of domains mediating transcriptional activation and cytoplasmic export in the caudal homeobox protein Cdx-3. J Biol Chem, 1999, 274 (9): 6011-6019

42    Olsen WA, Lloyd M, Korsmo H, He YZ. Regulation of sucrase and lactase in Caco-2 cells: Relationship to nuclear factors SIF-1 and NF-LPH. Am J Physiol, 1996, 271(4 Pt 1): G707-G713