Background
Materials and methods
Purine-motif triplex DNA formation and 33P-labeling
Electrophoretic mobility shift assay (EMSA) and super-shift EMSA
Statistical correlations
Purification of triplex DNA-binding proteins using biotin/streptavidin affinity
Western blotting
Reverse phase protein array (RPPA)
Results
Colorectal tumors have higher triplex DNA-binding activity than corresponding normal tissue
Tumor characteristics | Absolute (n = 63) | Relative (%) | |
---|---|---|---|
Sex | Male | 46 | 73% |
Female | 17 | 27% | |
Localization | Colon | 36 | 57.1% |
Rectum | 27 | 42.9% | |
TNM Staging | pT1 | 10 | 15.9% |
pT2 | 5 | 7.9% | |
pT3 | 34 | 54% | |
pT4 | 16 | 25.4% | |
Lymph Node Status | pN0 | 37 | 58.7% |
pN1 | 16 | 25.4% | |
pN2 | 10 | 15.9% | |
Metastasis Staging | M0 | 42 | 66.7% |
M1 | 21 | 33.3% | |
Status | alive | 43 | 68.1% |
dead | 20 | 31.7% |
Increased triplex DNA-binding activity in colorectal tumors correlates with lymph node disease, metastasis, and overall survival
(a) Grouping Variable: presence of disease in lymph nodes (N Stage) dichotomized | |||
---|---|---|---|
Ratio T/N | Ratio T/N | Ratio T/N | |
cytoplasm | nucleus | total | |
Mann-Whitney U
|
322.000
|
313.000
|
310.500
|
Wilcoxon W
|
1025.000
|
1016.000
|
1013.500
|
Z
|
-2.220
|
-2.345
|
-2.380
|
Asymp. Sig (2-tailed)
|
0.026
|
0.019
|
0.017
|
(b) Grouping Variable: presence of metastasis in distant organs (distant metastasis)
| |||
Cytoplasm tumor
|
Nucleus tumor
|
Total tumor
| |
Mann-Whitney U
|
342.500
|
309.500
|
321.000
|
Wilcoxon W
|
1245.500
|
1212.500
|
1224.000
|
Z
|
-1.689
|
-2.156
|
-1.993
|
Asymp. Sig (2-tailed)
|
0.091
|
0.031
|
0.046
|
Identification of U2AF65 as the protein present in the EMSA H3 complex
U2AF65 expression correlates with EMSA H3 values and p54nrb and PSF expression in tumor tissues and with a higher tumor stage
3BP expression correlated | U2AF65 | p54nrb | PSF |
---|---|---|---|
With EMSA H3 | |||
Normal cytoplasm
|
0.0028
|
0.91
|
0.66
|
Normal nucleus
|
0.015
|
0.11
|
0.075
|
Normal total
|
0.016
|
0.26
|
0.095
|
Tumor cytoplasm
|
1.8e-08
|
0.53
|
0.019
|
Tumor nucleus
|
5.9e-05
|
0.0071
|
0.036
|
Tumor total
|
1.8e-08
|
0.0048
|
7.5e-05
|
Correlation to U2AF65
|
p54nrb
|
PSF
| |
Normal cytoplasm
|
0.00066
|
0.085
| |
Normal nucleus
|
0.00037
|
0.073
| |
Normal total
|
0.00040
|
0.094
| |
Tumor cytoplasm
|
0.0041
|
0.0002
| |
Tumor nucleus
|
1e-06
|
0.0005
| |
Tumor total
|
1e-06
|
1e-06
|
Expression of the WRN helicase correlates with EMSA H3 binding activity
Reverse-phase protein array and western blot analysis of tissue extracts show a correlation of U2AF65 expression with total and truncated beta-catenin expression
EMSAH3 | U2AF65 | p54nrb | PSF | |
---|---|---|---|---|
Ph-Erk
|
**
| |||
NF-κB p65
|
***
|
***
| ||
Cyclin D1
|
***
|
***
| ||
GSK3β
|
***
|
*
| ||
c-myc
|
**
|
***
| ||
JNK1
|
***
|
***
| ||
PCNA
|
***
|
***
|
**
| |
β-catenin
|
***
|
***
| ||
Ph-Raf
|
**
| |||
Src
|
**
|
**
| ||
p 38 α
|
**
|
**
|
***
|
***
|
Cdk4
|
**
|
***
| ||
Akt1
|
**
|
***
| ||
ErbB1
|
***
|
***
|
**
|
***
|
Bcl-2
|
**
| |||
mTor
|
***
|
***
|
***
|
***
|
P13K p110 α
|
**
|
***
| ||
PLC γ
|
***
| |||
PTEN
|
***
|
***
|
***
|
***
|
Stat3
|
*
|
**
|
***
| |
Stat5
|
***
|
***
|
***
|
***
|