Figure 1.
Expression of DNMT3B is higher in RKO cells.
A. Western blot analysis of whole cell extracts from RKO and HCT (wild type and mutant) cells with antibodies specific for DNMTs. Affinity purified antibodies against DNMT3B were used for immunoblotting. B. Real-time RT-PCR analysis of DNMTs in RKO cells was performed using SYBR Green method. Data are mean of triplicate assays. Single and double asterisks denote p values ≤0.05 and ≤0.01, respectively. C. Ethidium bromide staining of DNA immunoprecipitated by DNMT3B antibody and input DNA that were used for CpG island microarray analysis.
Table 1.
Methylted DNMT3B Target Genes in RKO cells identified by ChIP on Chip.
Figure 2.
ChIP-CHOP assay to demonstrate association of DNMT3A and DNMT3B with the selected target genes.
A. Schematic diagram of ChIP-CHOP assay. B. Formaldehyde-cross-linked chromatin from RKO cells was immunoprecipitated with anti-DNMT3B or pre-immune IgG. DNAs pulled down were divided into three aliquots, which were either undigested or digested with Hpa II, Msp I and subjected to PCR with primers specific for CGIs of different genes that harbor Hpa II/Msp I sites. PCR products were resolved by agarose gel electrophoresis. Generation of PCR product in Hpa II digested DNA indicates methylation. U, H and M indicate PCR products obtained in mock-, Hpa II- digested and Msp I-digested DNA, respectively. Lack of PCR product with Msp I digested DNA confirmed complete digestion. C. Activation of several DNMT3B target genes in RKO cells after treatment with decitabine. RKO cells were treated with the drug for the indicate time periods. RNA from these cells was subjected to RT-PCR with gene-specific primers. Three hundred ng of cDNA for the test gene and 1 ng of cDNA were used for 18S rRNA.
Figure 3.
Analysis of methylation status of CGI of selected genes by COBRA in primary human colorectal tumors.
Location of the CGI of each gene as appeared in UCSC genome browser is depicted in the accompanied schematic diagrams. Bisulfite converted genomic DNA was amplified with gene specific primers followed by digestion with a methylation sensitive enzyme Taq I or BstU I. T and N denote tumor and matching normal, respectively. Sample numbers shown in red identify tumors with gene-specific methylation.
Figure 4.
Methylation of the CpGs spanning upstream and promoter regions of HOXB13 gene in primary colorectal tumors and colon cancer cell lines by MassARRAY.
A and B. Potential methylatable CpGs in these regions are shown after bisulfite conversion. CpGs sequenced by MassARRAY are boxed and numbered. It is notable that this technique provides average methylation status of CpGs that are in close proximity (numbered together as 1, 2 etc). Gray bars indicate samples that could not be sequenced. C.i. HEAT map of methylation profile of CpGs located within upstream CGI and promoter (TSS) region. The same amplicon of methylation density ranging from 0 to 100% was used to generate standard curve. C.ii. Box plot of quantitative analysis of methylation density in the upstream CGI and promoter regions in primary colorectal tumors and normals. Significance was assessed by Welch test (adaptation of t test, parametric, unequal variance, one-tailed). D. Upstream region (−5.4 kb) activates HOXB13 promoter activity in colon cancer cells. HOXB13 promoter regions (−1.2 kb and −5.4 kb) cloned into pGL3 basic vector (RLU1) were transfected into HCT116 cells along with internal control pRLTK (RLU2), followed by treatment with 10 nM estradiol (E) in phenol red free medium containing 5% charcoal stripped serum for different time periods. E. Only the upstream CGI is methylated in HCT cells, which undergoes site-specific and global demethylation by DNMT1 alone and both DNMT1, DNMT3B, respectively. i) MassARRAY, ii) real-time RT-PCR analysis of HOXB13, and iii) RT-PCR analysis of ERα and GAPDH.
Figure 5.
HOXB13 and TBX18 demonstrate anti-tumorignic property in colon cancer cells in vitro.
A. Western blot analysis of cell extracts prepared from RKO cells expressing Flag-tagged HOXB13 or TBX18. Analysis of cell growth by MTT assay (B), replication potential by 3H1-thymidine incorporation (C), clonogenic survival (D) of RKO cells (pool and a clone selected at random). Each assay was performed in triplicate. Single and double asterisks denote p values ≤0.05 and ≤0.01, respectively.
Figure 6.
HOXB13 but not TBX18 inhibited ability of RKO cells to form tumor in nude mice.
One million cells in PBS (100 µl) were injected into the flanks of nude mice and tumor size was monitored every week using a caliper. After 25 days tumors were excised and their weights were documented. A. Time course of the progression of tumor growth. B. Photograph of the tumors developed by vector-transfected, HoxB13 or TBX18 expressing cells. C. Tumor weight at the time of sacrifice. D. Western blot analysis of ectopic TBX-18 in the tumors T1 to T4 (shown in A) with anti-Flag antibody.