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DNA end binding activity and Ku70/80 heterodimer expression in human colorectal tumor
Paola Mazzarelli1, Paola Parrella, Davide Seripa
1Laboratory of Molecular Medicine and Biotechnology, Università Campus Bio-Medico, Via Longoni, 83, Rome 00155, Italy.
Aim:
To determine the DNA binding activity and protein levels of the Ku70/80 heterodimer, the functional mediator of the NHEJ activity, in human colorectal carcinogenesis.
Methods:
The Ku70/80 DNA-binding activity was determined by electrophoretic mobility shift assays in 20 colon adenoma and 15 colorectal cancer samples as well as matched normal colonic tissues. Nuclear and cytoplasmic protein expression was determined by immunohistochemistry and Western blot analysis.
Results:
A statistically significant difference was found in both adenomas and carcinomas as compared to matched normal colonic mucosa (P<0.00). However, changes in binding activity were not homogenous with approximately 50% of the tumors showing a clear increase in the binding activity, 30% displaying a modest increase and 15% showing a decrease of the activity. Tumors, with increased DNA-binding activity, also showed a statistically significant increase in Ku70 and Ku86 nuclear expression, as determined by Western blot and immunohistochemical analyses (P<0.001). Cytoplasmic protein expression was found in pathological samples, but not in normal tissues either from tumor patients or from healthy subjects.
Conclusion:
Overall, our DNA-binding activity and protein level are consistent with a substantial activation of the NHEJ pathway in colorectal tumors. Since the NHEJ is an error prone mechanism, its abnormal activation can result in chromosomal instability and ultimately lead to tumorigenesis.
Insights
Colorectal tumors show increased DNA binding activity and protein levels of Ku70/80, a key factor in non-homologous end joining (NHEJ) DNA repair. This activation may drive chromosomal instability and cancer development.
Area of Science:
- Molecular biology
- Cancer research
- Genetics
Background:
- The Ku70/80 heterodimer is crucial for non-homologous end joining (NHEJ), a DNA repair pathway.
- Aberrant DNA repair mechanisms are implicated in cancer development.
Purpose of the Study:
- To investigate the DNA binding activity and protein levels of the Ku70/80 heterodimer in human colorectal carcinogenesis.
- To understand the role of NHEJ pathway activation in colorectal tumor formation.
Main Methods:
- Electrophoretic mobility shift assays were used to determine Ku70/80 DNA-binding activity.
- Immunohistochemistry and Western blot analysis assessed nuclear and cytoplasmic protein expression.
- Samples included human colon adenomas, colorectal cancers, and matched normal colonic tissues.
Main Results:
- Ku70/80 DNA-binding activity and nuclear protein expression were significantly increased in both adenomas and carcinomas compared to normal tissues.
- Approximately 50% of tumors showed a clear increase in binding activity, with others showing modest increases or decreases.
- Cytoplasmic expression of Ku70/80 was observed in pathological samples but not in normal tissues.
Conclusions:
- The findings suggest substantial activation of the NHEJ pathway in colorectal tumors.
- The error-prone nature of NHEJ, when abnormally activated, can contribute to chromosomal instability and tumorigenesis.
- Ku70/80 activity and expression levels serve as potential biomarkers in colorectal cancer.
