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Induction of intestinalization in human esophageal keratinocytes is a multistep process - PubMed

Induction of intestinalization in human esophageal keratinocytes is a multistep process

Jianping Kong et al. Carcinogenesis. 2009 Jan.

Abstract

Barrett's esophagus (BE) is the replacement of normal squamous esophageal mucosa with an intestinalized columnar epithelium. The molecular mechanisms underlying its development are not understood. Cdx2 is an intestine-specific transcription factor that is ectopically expressed in BE, but its role in this process is unclear. Herein, we describe a novel cell culture model for BE. Retroviral-mediated Cdx2 expression in immortalized human esophageal keratinocytes [EPC-human telomerase reverse transcriptase (hTERT)] could transiently be established but not maintained and was associated with a reduction in cell proliferation. Coexpression of cyclin D1, but not a dominant-negative p53, rescued proliferation in the Cdx2-expressing cells. Cdx2 expression in the EPC-hTERT.D1 cells decreased cell proliferation but did not induce intestinalization. We investigated for other treatments to enhance intestinalization and found that acidic culture conditions uniformly killed EPC-hTERT.D1.Cdx2 cells. However, treatment with 5-aza-2-deoxycytidine (5-AzaC) to demethylate epigenetically silenced genes did appear to be tolerated. Multiple Cdx2 target genes, markers of intestinal differentiation and markers of BE, were induced by this 5-AzaC treatment. More interestingly, the expression level of several of these genes was enhanced only in the EPC-hTERT.D1-Cdx2 cells treated with 5-AzaC. Two of these, SLC26a3/DRA (downregulated in adenoma) and Na+/H+ exchanger 2 (NHE2), were not previously known to be elevated in BE; however, we confirmed their elevation in BE tissue samples. 5-AzaC treatment also induced cell senescence, even at low doses. We conclude that ectopic proliferation signals, alterations in epigenetic gene regulation and the inhibition of tumor suppressor mechanisms are required for Cdx2-mediated intestinalization of human esophageal keratinocytes in BE.

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Figures

Fig. 1.
Fig. 1.

Cdx2 expression is not sustained in normal EPC-hTERT keratinocytes. Human EPC-hTERT keratinocytes were infected with a retroviral vector to induce Cdx2 expression (MIGR-Cdx2) or the control empty viral vector MIGR1. GFP serves as a marker for viral infection and gene expression. (A) Fluorescent image for GFP+ cells overlaid with phase contrast image. At 72 h, nearly equivalent numbers of GFP+ cells with both vectors. After 10 days in culture, repeat images demonstrate highly significant reduction in GFP+ expression in those cells infected with the MIGR-Cdx2 virus. (B) The number of GFP+ cells per total cells in six high power fields (HPFs) was determined on days 2, 3, 5, 7, 8 and 10 post-retroviral infection. Averages and standard deviations were calculated and graphed. Black circle, EPC-hTERT.MIGR. Black square, EPC-hTERT.Cdx2. a, significantly differs from MIGR same day controls using analysis of variance and Tukey Rank Mean testing, P < 0.005. b, significantly differs from day 2 MIGR controls, P < 0.005.

Fig. 2.
Fig. 2.

Cdx2 expression in normal keratinocytes requires exogenous stimulus for proliferation. (A) Western blot analysis demonstrating doxycyclin-regulated cyclin D1 protein expression. Enhanced cyclin D1 protein levels were noted in EPC-hTERT.D1 (EPC.D1) but not control cells (EPC.P) in the absence of doxycyclin. However, doxycyclin (1 μg/ml) in the cell culture medium suppressed this expression. (B) Western blot results illustrating overexpression of Cdx2 in EPC-hTERT.D1-Cdx2 cells but not the EPC-hTERT.D1-MIGR1 control cells. (C) Cdx2 expression in the EPC-hTERT.D1 cells reduces proliferation. [H3]Thymidine incorporation assay in the absence (light gray bars) and presence of doxycyclin (1 μg/ml) (dark gray bars). In the absence of exogenous cyclin D1, Cdx2 expression is associated with a significant reduction in [H3]thymidine incorporation when compared with controls. Averages and standard deviations were calculated (n = 3, P < 0.05). (D) WST-1 cell accumulation studies performed on EPC-hTERT.D1-MIGR1 (EM) and EPC-hTERT.D1-Cdx2 (EX2) cells in the absence or presence of doxycycline (D; 1 μg/ml). There was a significant decrease in the accumulation of EPC-hTERT.D1-Cdx2 cells compared with MIGR1 control cells in the presence or absence of exogenous cyclin D1. Averages and standard deviations from eight separate wells for each treatment group were calculated and graphed. One of three experiments is shown.

Fig. 3.
Fig. 3.

A dominant-negative p53 does not rescue Cdx2 expression in EPC-hTERT keratinocytes. EPC-hTERT keratinocytes expressing a dominant-negative p53 were established using the pBabe-puro-p53R175H vector to direct expression of the p53R175H mutant. (A) Western blot of p53 protein levels in EPC-hTERT cells receiving the empty pBabe control vector (EPC.P) or the pBabe-puro-p53R175H (EPC.p53R175H). The blot was reprobed for actin levels as loading control. (B) As before, fluorescent image for GFP+ cells overlaid with phase contrast image. EPC.P or EPC.p53R175H cells were subsequently infected with the MIGR-Cdx2 virus to induce Cdx2 expression (EPC.P.X2 and E.p53.X2) or the MIGR1 empty control vector (EPC.P.M and E.p53.M). Photodocumentation was obtained at 3 and 9 days post-infection with the MIGR-based vectors. At 3 days, nearly equivalent numbers of GFP+ cells with both vectors. By day 9, there is a highly significant reduction in GFP+ expression in those cells infected with the MIGR-Cdx2 virus. (C) The number of GFP+ cells per total cells in six high-power fields (HPFs) was determined on days 2, 4, 6, 8 and 9 post-retroviral infection. Averages and standard deviations were calculated and graphed. Black X, dashed line: EPC.P.M. White triangle, solid line: EPC.P.X2. White square, dashed line: E.p53.M. Black diamond, solid line: E.p53.X2. a, significantly differs from day 9 MIGR controls using analysis of variance and Tukey Rank Mean testing, P < 0.05.

Fig. 4.
Fig. 4.

Cdx2 expression alone has minimal effects on EPC-hTERT intestinal transdifferentiation. (A and B) Quantitative SYBR-green RT–PCR analysis of gene expression in EPC-hTERT.D1-MIGR1 (EPC.D1.M) or EPC-hTERT.D1-Cdx2 (EPC.D1.X2) cells treated for 5 days with 5 μM of 5-azacytidine (5AZA) or diluent control (CTR). Genes tested included Claudin2, CEACAM6, Villin, LPH, CAI, RELM-β, NHE2, KRT20 and DRA/SLC26A3. The PCR control was the phosphoprotein 36B4. ΔCt values were calculated after duplicate PCRs for each sample, then statistical analysis was performed (analysis of variance and Tukey Rank Mean). ΔΔCt values were then calculated and used to determine fold change in expression (n = 6 samples). Black circle = significantly differs from MIGR-diluent control, P < 0.0005; black star = significantly differs from MIGR-diluent control, P < 0.005. (C) Quantitative SYBR-green RT–PCR analysis of CDX1 and CDX2 gene expression in EPC-hTERT.D1.MIGR1 (EPC.D1.M) or EPC-hTERT.D1.Cdx2 (EPC.D1.X2) cells treated with 5 μM 5-azacytidine (5AzaC) or diluent control. PCR controls and the calculation of average ΔΔCt values (and standard deviations) were carried out as before (n = 3). (D) Western blot for CDX1, CDX2, DRA/SLC26a3, villin and NHE2 protein levels in EPC-hTERT.D1.MIGR (MIGR1) or EPC-hTERT.D1.Cdx2 cells (CDX2). Cells were treated with 5 μM 5-azacytidine (5AzaC) or diluent control. The blots were routinely stripped and reprobed for actin levels as loading control. One of three experiments is shown. (E) Quantitative densitometry measurements of selected western band densities. Values were normalized to actin bands, then expressed as fold change compared with EPC-hTERT.D1.MIGR cells treated with diluent (white bars). Black bars, EPC-hTERT.D1.MIGR cells treated with 5 μM 5-azacytidine. Light gray bars, EPC-hTERT.D1.Cdx2 cells with diluent. Dark gray bars, EPC-hTERT.D1.Cdx2 cells with 5 μM 5-azacytidine.

Fig. 5.
Fig. 5.

Intestine-associated genes NHE2 and SLC26a3 are induced in human BE. (A) Quantitative real-time PCR measurement of NHE2, DRA/SLC2A3, CAI and KRT20 mRNA levels in biopsies from human BE and adjacent normal esophageal mucosa. (B) Representative immunohistochemical staining for DRA/SLC26A3 and NHE2 protein in BE biopsy specimen. Normal colon and normal esophagus are presented as controls. One of several Barrett’s biopsy specimens is shown.

Fig. 6.
Fig. 6.

Senescence checkpoint genes are activated by chromatin remodeling agents 5-AzaC in esophageal keratinocytes. After 5-AzaC treatment, EPC-hTERT.D1-Cdx2 and MIGR1 (EPC.D1.X2 and EPC.D1.M, respectively) control cells exhibit characteristics of senescence including altered morphology, increased cell volume and subconfluent growth arrest in the normal medium. (A) Morphology of EPC-hTERT.D1.Cdx2 and control EPC-hTERT.D1.MIGR1 cells after 5AzaC at 1 and 5 μM for 5 days. Induction of senescence-associated β-galactosidase activity was identified by staining (blue). (B) 5-AzaC treatment induces the expression of senescence-associated p16(INK4a) and p21(Waf1/Cip1). Western Blot for p16 and p21 after cells were treated with 1 μM 5-AzaC or control diluent for 5 days. β-Actin served as an internal loading control. (C) Proposed model for the early molecular events preceding the onset of intestinalization in esophageal keratinocytes.

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