DKK1 expression represses Wnt pathway activation in MM.

<p>(A) MM cell lines OPM-1 and UM-1 were transduced with either the LZRS-pBMN-IRES-eGFP (control) or the LZRS-pBMN-DKK1-IRES-eGFP (DKK1) virus. Conditioned medium of sorted, transduced cells was harvested and immunoblotted using a goat polyclonal antibody against DKK1. Representative immunoblot confirms the expression of DKK1 in the conditioned medium of LZRS-pBMN-DKK1-IRES-eGFP transduced cells. β-actin is shown as internal control for equal cell number. (B) Cytoplasmic and nuclear proteins were prepared from the LZRS-pBMN-IRES-eGFP (control) or the LZRS-pBMN-DKK1-IRES-eGFP (DKK1) MM cells, stimulated for 24h with Wnt3a conditioned medium (+). As a control, L-cells conditioned medium was applied (−).To assess β-catenin accumulation, nuclear and cytoplasmic cells lysate was immunoblotted by using a monoclonal anti-β-catenin antibody. The bottom part of the blot was stained with β-tubulin and Histone H2B as controls for cytoplasmic and nuclear proteins, respectively. (C) LZRS-pBMN-IRES-eGFP (control) or the LZRS-pBMN-DKK1-IRES-eGFP (DKK1) cells were transfected with TOPFLASH reporter and renilla contruct. 24 hours upon transfection cells were treated with L-cells conditioned medium (−) or Wnt3a conditioned medium (+).The relative light units value of LZRS-pBMN-IRES-eGFP cells treated with L-cells conditioned medium was normalized to 1. The mean ± SD of representative experiment performed in triplicate is shown. * indicates p value<0.05 *** indicates p value<0.001. by student's t test.</p

<i>DKK1</i> promoter methylation in MM cell lines.

<p>(A) Total RNA was isolated and RT–PCR analyses were performed with the specific primers indicated. Complementary DNA from prostate cancer cell line (PC-3) was used as positive control (PC) for DKK1 expression. The β-actin expression was used as a loading control. (B) Schematic representation of the promoter area analyzed for <i>DKK1</i>, containing a CpG island. White arrows indicate the positions of primers used for bisulfite sequencing, and black arrows indicate the positions of primers used for methylation specific PCR. Each of the CpG dinucleotides is presented as open circle. (C) <i>Upper panel</i>. Representation of bisulfite genomic sequencing results of 5 clones of the <i>DKK1</i> promoter region in HT-29 and DLD-1 colon cell lines used as unmethylated (U_DNA) and methylated (M_DNA) control, respectively. The amplified 326 bp product corresponds to the <i>DKK1</i> promoter region from −193 to +122. In total, 18 CpG dinucleotides (CpGs) within the CpG island were analyzed and are represented as open and closed circles, which indicate unmethylated and methylated CpG sites, respectively. <i>Lower panel</i>. Electropherograms of bisulfite modified DNA from <i>DKK1</i> CpG island in HT-29 (U_DNA) and DLD-1 (M_DNA) cells. (D) Methylation specific PCR of the CpG island of the <i>DKK1</i> promoter region in MM cell lines. DNA bands in lanes labeled with U indicate PCR products amplified with primers recognizing unmethylated promoter sequences, whereas DNA bands in lanes labeled with M represent amplified products with primers designed for the methylated template. (E) Bisulfite sequencing analysis of the the <i>DKK1</i> promoter region in multiple myeloma cell lines, open circles indicating unmethylated CpG sites, and closed circles representing methylated CpG sites. Percentages indicate the fraction of methylated CpG dinucleotides of the total CpG sites analyzed.</p

Analysis of <i>DKK1</i> promoter methylation in MM bone marrow samples.

<p>Methylation specific PCR of the CpG island of the <i>DKK1</i> promoter region in the bone marrow samples of twelve patients with multiple myeloma (P1–P12), HT-29 and DLD-1 colon cell lines were used as unmethylated (U_DNA) and methylated (M_DNA) control respectively. DNA bands in lanes labeled with U and M indicate PCR products amplified with primers recognizing unmethylated and methylated promoter sequences respectively.</p

Restoration of <i>DKK1</i> expression in MM cell lines by 5-aza-2-deoxycytidine treatment.

<p>(A) <i>DKK-1</i> promoter methylation analyzed by bisulfite genomic sequencing of 10 clones, on DNA isolated from 5-aza-2-deoxycytidine treated (5-aza-CdR) and untreated (PBS) MM cell lines UM-1 and OPM-1. Frequency of methylation was calculated by dividing the number of methylated CpG sites by the total number of analyzed CpG sites. (B) Reverse transcriptase-PCR analysis for DKK1 gene expression in multiple myeloma cell lines in the absence and presence of the demethylating agent 5-aza-2-deoxycytidine. β-actin expression is shown as an input control.</p

The relation between the Wnt pathway activation and the loss of DKK1 expression during MM progression.

<p>(A) Representative pictures of immunohistochemical staining of a multiple myeloma patient displaying either high DKK-1 and low β-catenin expression (upper panel) or with DKK-1 loss and increased nuclear β-catenin localization (lower panel). Immunohistochemical stainings are shown for CD138 (left column), β-catenin (middle column) and DKK1 (right column). (B) Nuclear β-catenin expression in relation to multiple myeloma progression (n = 48, p<0.05). (C) DKK-1 expression in relation to multiple myeloma progression (p<0.05). (D) Representative pictures of immunocytochemical staining of multiple myeloma cell lines with goat polyclonal anti-DKK1 antibody (magnification: 400×). Prostate cancer cell line (PC-3) was used as positive control (PC) for the DKK-1 staining. (E) Relation between the loss of DKK-1 expression and nuclear localization of β-catenin (p>0.05). A significant correlation (p<0.05) between expression of nuclear β-catenin and DKK-1 was observed in the two extreme groups identified based on β-catenin expression. * indicates p value<0.05.</p