The online version of this article (doi:10.1186/1476-4598-11-38) contains supplementary material, which is available to authorized users.
The authors declare that they have no competing interests.
LDN, HM, MWVD and HA designed the study; CB, DB, PK, and GM performed all statistical analysis and collected patient clinical data; LDN and HM performed all experiments; UK supervised reverse phase protein array experiments; LDN, DPH, MWVD and HA wrote the manuscript. All authors read and approved the final manuscript.
Tri- and tetra-nucleotide repeats in mammalian genomes can induce formation of alternative non-B DNA structures such as triplexes and guanine (G)-quadruplexes. These structures can induce mutagenesis, chromosomal translocations and genomic instability. We wanted to determine if proteins that bind triplex DNA structures are quantitatively or qualitatively different between colorectal tumor and adjacent normal tissue and if this binding activity correlates with patient clinical characteristics.
Extracts from 63 human colorectal tumor and adjacent normal tissues were examined by gel shifts (EMSA) for triplex DNA-binding proteins, which were correlated with clinicopathological tumor characteristics using the Mann-Whitney U, Spearman’s rho, Kaplan-Meier and Mantel-Cox log-rank tests. Biotinylated triplex DNA and streptavidin agarose affinity binding were used to purify triplex-binding proteins in RKO cells. Western blotting and reverse-phase protein array were used to measure protein expression in tissue extracts.
Increased triplex DNA-binding activity in tumor extracts correlated significantly with lymphatic disease, metastasis, and reduced overall survival. We identified three multifunctional splicing factors with biotinylated triplex DNA affinity: U2AF65 in cytoplasmic extracts, and PSF and p54nrb in nuclear extracts. Super-shift EMSA with anti-U2AF65 antibodies produced a shifted band of the major EMSA H3 complex, identifying U2AF65 as the protein present in the major EMSA band. U2AF65 expression correlated significantly with EMSA H3 values in all extracts and was higher in extracts from Stage III/IV vs. Stage I/II colon tumors (p = 0.024). EMSA H3 values and U2AF65 expression also correlated significantly with GSK3 beta, beta-catenin, and NF- B p65 expression, whereas p54nrb and PSF expression correlated with c-Myc, cyclin D1, and CDK4. EMSA values and expression of all three splicing factors correlated with ErbB1, mTOR, PTEN, and Stat5. Western blots confirmed that full-length and truncated beta-catenin expression correlated with U2AF65 expression in tumor extracts.
Increased triplex DNA-binding activity in vitro correlates with lymph node disease, metastasis, and reduced overall survival in colorectal cancer, and increased U2AF65 expression is associated with total and truncated beta-catenin expression in high-stage colorectal tumors.
Additional file 1: Figure S1. Electrophoretic Mobility Shift Assay (EMSA) of patient tissue lysates and HeLa nuclear extract with triplex and parent duplex DNA probes. 33P‐labeled purine‐motif duplex or triplex DNA (1 nM) was complexed with 5 μg protein from normal tissue cytoplasmic (N cy), normal nuclear (N nu), tumor tissue cytoplasmic (T cy) or tumor nuclear (T nu) extracts of colorectal cancer patients. 1.25 μg HeLa nuclear extract (H) was used as a control in lanes 6 and 12. Purine triplex probe alone is in lane 1 and duplex probe alone is in lane 7. Figure S2a. Electrophoretic Mobility Shift Assay (EMSA) of Cytoplasmic and Nuclear Extracts from Eight Colorectal Cancer Cell Lines with Purine triplex DNA. 33P‐labeled purine‐motif triplex DNA (1 nM) was complexed with 1.25 μg total protein from cytoplasmic (cy) or nuclear (nuc) extracts from eight colorectal cancer cell lines. 1.25 μg HeLa cytoplasmic and nuclear extracts were used as positive (+) controls. Each reaction also contained 2 μg poly (dI‐dC) carrier DNA. The purine triplex DNA probe alone is shown in lane 1. Figure S2b. Western blots showing expression of three candidate triplex DNA‐binding proteins in eight colorectal cancer cell lines. Total protein (25 μg) from cytoplasmic (cy) and nuclear (nu) extracts from eight colorectal cancer cell lines were separated using 10% SDS‐PAGE and electro‐transferred to nitrocellulose membranes. Blots were incubated with the antibodies against PSF, U2AF65, p54nrb, beta‐catenin, and actin, then the appropriate secondary antibody and detected using chemiluminescence and autoradiography. Figure S3. Lack of a super‐shifted H3 band in RKO nuclear extract by super‐shift EMSA with antibodies against PSF and p54nrb. 33P‐labeled triplex DNA (1 nM) was complexed with 1.5 μg total protein from RKO nuclear extracts (lanes 2‐9). Lane 1, triplex DNA probe alone; Lane 2, no antibody; lane 3, 400 ng anti‐U2AF65 antibody MC3; lane 4, 1000 ng anti‐U2AF65 antibody MC3; lane 5, 400 ng anti‐PSF antibody; lane 6 1000 ng anti‐PSF antibody; lane 7, 400 ng anti‐p54nrb antibody; lane 8, 1000 ng anti‐p54nrb antibody; lane 9, mouse IgG antibody (negative control). Each reaction also contained 2 μg poly (dI‐dC) carrier DNA. Figure S4. Quantitation of Protein Expression of PSF, U2AF65, p54nrb, and beta‐catenin obtained from six colorectal cancer patients’ tissue extracts. Autoradiographs from Western blots in Figure 6 were scanned, and protein expression bands were quantitated using NIH Image J. Protein expression was normalized by dividing by the samples’ corresponding actin value and graphed using Graph Pad. Figure S5. Beta‐catenin Expression by Tumor type and Stage. Western blots using an anti‐beta‐catenin antibody to examine expression in patient extracts were described for Figure 6. Beta‐catenin expression values were normalized by dividing the actin expression value in each extract, and plotted according to colon or rectum tumor stage using the R program. N cyto, cytoplasmic normal tissue extracts; N nuc, nuclear normal tissue extracts; T cyto, cytoplasmic tumor tissue extracts; T nuc, nuclear tumor tissue extracts. (PDF 610 KB)12943_2011_1058_MOESM1_ESM.pdf
Additional file 2: DB-Triplexdata. (RTF 490 KB)12943_2011_1058_MOESM2_ESM.rtf
Additional file 3: PK Statistical analysis Triplex. (PDF 597 KB)12943_2011_1058_MOESM3_ESM.pdf
Additional file 4: DBuergy Correlations(1). (XLS 66 KB)12943_2011_1058_MOESM4_ESM.xls
Additional file 5: Daniel Apr 5(1). (XLS 42 KB)12943_2011_1058_MOESM5_ESM.xls
Additional file 6: histograms_proteins_groups. (PDF 191 KB)12943_2011_1058_MOESM6_ESM.pdf
Additional file 7: Table S1. RPPA antibodies and Spearman correlation p values. (PDF 114 KB)12943_2011_1058_MOESM7_ESM.pdf
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- Triplex DNA-binding proteins are associated with clinical outcomes revealed by proteomic measurements in patients with colorectal cancer
Laura D Nelson
Michael W Van Dyke
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