A genetic screen using a library of 6961 siRNAs led to the identification of SHP1 (PTPN6), a tumor suppressor frequently mutated in malignant lymphomas, leukemias and prostate cancer, as a potential synthetic lethal partner of the DNA repair protein polynucleotide kinase/phosphatase (PNKP). After confirming the partnership with SHP-1, we observed that co-depletion of PNKP and SHP-1 induced apoptosis. A T-cell lymphoma cell line that is SHP-1-deficient (Karpas 299) was shown to be sensitive to a chemical inhibitor of PNKP, but resistance was restored by expression of wild-type SHP-1 in these cells. We determined that while SHP-1 depletion does not significantly impact DNA strand-break repair, it does amplify the level of reactive oxygen species (ROS) and elevate endogenous DNA damage. The ROS scavenger WR1065 afforded protection to SHP-1 depleted cells treated with the PNKP inhibitor. We propose that co-disruption of SHP-1 and PNKP leads to an increase in DNA damage that escapes repair, resulting in the accumulation of cytotoxic double-strand breaks and induction of apoptosis. This supports an alternative paradigm for synthetic lethal partnerships that could be exploited therapeutically.
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[Show abstract][Hide abstract]ABSTRACT: A recent screen of 6961 siRNAs to discover possible synthetic lethal partners of the DNA repair protein polynucleotide kinase/phosphatase (PNKP) led to the identification of the potent tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Here we have confirmed the PNKP/PTEN synthetic lethal partnership in a variety of different cell lines including the PC3 prostate cancer cell line, which is naturally deficient in PTEN. We provide evidence that co-depletion of PTEN and PNKP induces apoptosis. In HCT116 colon cancer cells the loss of PTEN is accompanied by an increased background level of DNA double strand breaks, which accumulate in the presence of an inhibitor of PNKP DNA 3'-phosphatase activity. Complementation of PC3 cells with several well-characterized mutated PTEN cDNAs indicated that the critical function of PTEN required to prevent toxicity induced by an inhibitor of PNKP is most likely associated with its cytoplasmic lipid phosphatase activity. Finally, we show that modest inhibition of PNKP in a PTEN knockout background enhances cellular radiosensitivity, suggesting that such a "synthetic sickness" approach involving the combination of PNKP inhibition with radiotherapy may be applicable to PTEN-deficient tumors.
Full-text · Article · Jul 2013 · Molecular Cancer Therapeutics
[Show abstract][Hide abstract]ABSTRACT: Synthetic lethal screen technology has been successfully introduced into the field of cancer research, and stands as a powerful method to reveal gene interactions for cancer related genes. The composition of the technology and its significant achievements in cancer related field are introduced here. Researches revealed a number of synthetic lethal genes for oncogene RAS, poly (ADP ribose) polymerase, MYC or tumor suppressor gene p53 and phosphatase and tensin homolog. Chemosensitizer genes for cytotoxic drugs such as paclitaxel and vinblastine were also reported. These researches provide to the mechanism of drug resistance and tumor recurrence, while promoting developments of molecular targeted agents combination therapy for cancer.
No preview · Article · Dec 2013 · Chinese Journal of Pharmacology and Toxicology
[Show abstract][Hide abstract]ABSTRACT: As two commonly used tool enzymes, DNA ligase and polynucleotide kinase/phosphatase (PNKP) play important roles in DNA metabolism. More and more studies show that regulation of their activity represents promising means for cancer therapy. To detect the activity of DNA ligase with high sensitivity and specificity, a G-quadruplex DNAzyme-based DNA ligase sensor was developed. In this sensor, the use of G-quadruplex DNAzyme eliminated the needs for any labeled oligonucleotide probes, thus making label-free detection possible. The introduction of rolling circle amplification (RCA) reaction could lead to the formation of multimeric G-quadruplexes containing thousands of G-quadruplex units, which can provide highly active hemin-binding sites, thus significantly improving the sensitivity of the sensor. The proposed sensor allowed specific detection of T4 DNA ligase with a detection limit of 0.0019U/mL. By adding a PNKP-triggered 5'-phosphroylation step of the template DNA, the above sensing strategy could be easily extended to the design of PNKP sensor. The established sensor allowed specific detection of T4 PNKP with a detection limit of 0.0018U/mL. In addition, these two sensors could also be used for the studies on inhibitors of these two enzymes.
No preview · Article · Dec 2013 · Biosensors & Bioelectronics