| 1. |
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| 2. |
- Bryant, Helen E, et al.
(författare)
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PARP is activated at stalled forks to mediate Mre11-dependent replication restart and recombination.
- 2009
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Ingår i: The EMBO journal. - : Wiley. - 1460-2075 .- 0261-4189. ; 28:17, s. 2601-15
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Tidskriftsartikel (refereegranskat)abstract
- If replication forks are perturbed, a multifaceted response including several DNA repair and cell cycle checkpoint pathways is activated to ensure faithful DNA replication. Here, we show that poly(ADP-ribose) polymerase 1 (PARP1) binds to and is activated by stalled replication forks that contain small gaps. PARP1 collaborates with Mre11 to promote replication fork restart after release from replication blocks, most likely by recruiting Mre11 to the replication fork to promote resection of DNA. Both PARP1 and PARP2 are required for hydroxyurea-induced homologous recombination to promote cell survival after replication blocks. Together, our data suggest that PARP1 and PARP2 detect disrupted replication forks and attract Mre11 for end processing that is required for subsequent recombination repair and restart of replication forks.
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| 3. |
- Gottipati, Ponnari, et al.
(författare)
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Poly(ADP-Ribose) Polymerase Is Hyperactivated in Homologous Recombination-Defective Cells
- 2010
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Ingår i: Cancer Research. - 0008-5472 .- 1538-7445. ; 70:13, s. 5389-5398
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Tidskriftsartikel (refereegranskat)abstract
- Poly(ADP-ribose) (PAR) polymerase 1 (PARP1) is activated by DNA single-strand breaks (SSB) or at stalled replication forks to facilitate DNA repair. Inhibitors of PARP efficiently kill breast, ovarian, or prostate tumors in patients carrying hereditary mutations in the homologous recombination (HR) genes BRCA1 or BRCA2 through synthetic lethality. Here, we surprisingly show that PARP1 is hyperactivated in replicating BRCA2-defective cells. PARP1 hyperactivation is explained by the defect in HR as shRNA depletion of RAD54, RAD52, BLM, WRN, and XRCC3 proteins, which we here show are all essential for efficient HR and also caused PARP hyperactivation and correlated with an increased sensitivity to PARP inhibitors. BRCA2-defective cells were not found to have increased levels of SSBs, and PAR polymers formed in HR-defective cells do not colocalize to replication protein A or gamma H2AX, excluding the possibility that PARP hyperactivity is due to increased SSB repair or PARP induced at damaged replication forks. Resistance to PARP inhibitors can occur through genetic reversion in the BRCA2 gene. Here, we report that PARP inhibitor-resistant BRCA2-mutant cells revert back to normal levels of PARP activity. We speculate that the reason for the sensitivity of HR-defective cells to PARP inhibitors is related to the hyperactivated PARP1 in these cells. Furthermore, the presence of PAR polymers can be used to identify HR-defective cells that are sensitive to PARP inhibitors, which may be potential biomarkers. Cancer Res; 70(13); 5389-98. (C) 2010 AACR.
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| 4. |
- Grinkevich, Vera V., et al.
(författare)
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Novel Allosteric Mechanism of Dual p53/MDM2 and p53/MDM4 Inhibition by a Small Molecule
- 2022
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media S.A.. - 2296-889X. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Restoration of the p53 tumor suppressor for personalised cancer therapy is a promising treatment strategy. However, several high-affinity MDM2 inhibitors have shown substantial side effects in clinical trials. Thus, elucidation of the molecular mechanisms of action of p53 reactivating molecules with alternative functional principle is of the utmost importance. Here, we report a discovery of a novel allosteric mechanism of p53 reactivation through targeting the p53 N-terminus which promotes inhibition of both p53/MDM2 (murine double minute 2) and p53/MDM4 interactions. Using biochemical assays and molecular docking, we identified the binding site of two p53 reactivating molecules, RITA (reactivation of p53 and induction of tumor cell apoptosis) and protoporphyrin IX (PpIX). Ion mobility-mass spectrometry revealed that the binding of RITA to serine 33 and serine 37 is responsible for inducing the allosteric shift in p53, which shields the MDM2 binding residues of p53 and prevents its interactions with MDM2 and MDM4. Our results point to an alternative mechanism of blocking p53 interaction with MDM2 and MDM4 and may pave the way for the development of novel allosteric inhibitors of p53/MDM2 and p53/MDM4 interactions.
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| 5. |
- Gubanova, Evgenia, et al.
(författare)
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Downregulation of SMG-1 in HPV-Positive Head and Neck Squamous Cell Carcinoma Due to Promoter Hypermethylation Correlates with Improved Survival
- 2012
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Ingår i: Clinical Cancer Research. - 1078-0432 .- 1557-3265. ; 18:5, s. 1257-1267
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: Human papillomavirus (HPV) is linked with a subset of head and neck squamous cell carcinomas (HNSCC). HPV-positive HNSCCs show a better prognosis than HPV-negative HNSCCs, which may be explained by sensitivity of the HPV-positive HNSCCs to ionizing radiation (IR). Although the molecular mechanism behind sensitivity to IR in HPV-positive HNSCCs is unresolved, DNA damage response (DDR) might be a significant determinant of IR sensitivity. An important player in the DDR, SMG-1 (suppressor with morphogenetic effect on genitalia), is a potential tumor suppressor and may therefore be deregulated in cancer. No studies have yet been conducted linking defects in SMG-1 expression with cancer. We investigated whether deregulation of SMG-1 could be responsible for defects in the DDR in oropharyngeal HNSCC. Experimental Design: Expression and promoter methylation status of SMG-1 were investigated in HNSCCs. To identify a functional link between HPV infection and SMG-1, we transfected the HPV-negative cells with an E6/E7 expression construct. SMG-1 short hairpin RNAs were expressed in HPV-negative cells to estimate survival upon IR. Results: Forced E6/E7 expression in HPV-negative cells resulted in SMG-1 promoter hypermethylation and decreased SMG-1 expression. Due to promoter hypermethylation, HPV-positive HNSCC cells and tumors express SMG-1 at lower levels than HPV-negative SCCs. Depletion of SMG-1 in HPV-negative HNSCC cells resulted in increased radiation sensitivity, whereas SMG-1 overexpression protected HPV-positive tumor cells from irradiation. Conclusions: Levels of SMG-1 expression negatively correlated with HPV status in cancer cell lines and tumors. Diminished SMG-1 expression may contribute to the enhanced response to therapy exhibited by HPV-positive HNSCCs.
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| 6. |
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| 7. |
- Gubanova, Evgenia, et al.
(författare)
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SMG-1 suppresses CDK2 and tumor growth by regulating both the p53 and Cdc25A signaling pathways
- 2013
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Ingår i: Cell Cycle. - : Informa UK Limited. - 1538-4101 .- 1551-4005. ; 12:24, s. 3770-3780
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Tidskriftsartikel (refereegranskat)abstract
- The DNA damage response is coordinated by phosphatidylinositol 3-kinase-related kinases, ATM, ATR, and DNA-PK. SMG-1 is the least studied stress-responsive member of this family. Here, we show that SMG-1 regulates the G 1/S checkpoint through both a p53-dependent, and a p53-independent pathway. We identify Cdc25A as a new SMG-1 substrate, and show that cells depleted of SMG-1 exhibit prolonged Cdc25A stability, failing to inactivate CDK2 in response to radiation. Given an increased tumor growth following depletion of SMG-1, our data demonstrate a novel role for SMG-1 in regulating Cdc25A and suppressing oncogenic CDK2 driven proliferation, confirming SMG-1 as a tumor suppressor.
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| 8. |
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| 9. |
- Issaeva, Natalia, et al.
(författare)
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6-Thioguanine Selectively Kills BRCA2-Defective Tumors and Overcomes PARP Inhibitor Resistance
- 2010
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Ingår i: Cancer Research. - 0008-5472 .- 1538-7445. ; 70:15, s. 6268-6276
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Tidskriftsartikel (refereegranskat)abstract
- Familial breast and ovarian cancers are often defective in homologous recombination (HR) due to mutations in the BRCA1 or BRCA2 genes. Cisplatin chemotherapy or poly(ADP-ribose) polymerase (PARP) inhibitors were tested for these tumors in clinical trials. In a screen for novel drugs that selectively kill BRCA2-defective cells, we identified 6-thioguanine (6TG), which induces DNA double-strand breaks (DSB) that are repaired by HR. Furthermore, we show that 6TG is as efficient as a PARP inhibitor in selectively killing BRCA2-defective tumors in a xenograft model. Spontaneous BRCA1-defective mammary tumors gain resistance to PARP inhibitors through increased P-glycoprotein expression. Here, we show that 6TG efficiently kills such BRCA1-defective PARP inhibitor-resistant tumors. We also show that 6TG could kill cells and tumors that have gained resistance to PARP inhibitors or cisplatin through genetic reversion of the BRCA2 gene. Although HR is reactivated in PARP inhibitor-resistant BRCA2-defective cells, it is not fully restored for the repair of 6TG-induced lesions. This is likely to be due to several recombinogenic lesions being formed after 6TG. We show that BRCA2 is also required for survival from mismatch repair-independent lesions formed by 6TG, which do not include DSBs. This suggests that HR is involved in the repair of 6TG-induced DSBs as well as mismatch repair-independent 6TG-induced DNA lesion. Altogether, our data show that 6TG efficiently kills BRCA2-defective tumors and suggest that 6TG may be effective in the treatment of advanced tumors that have developed resistance to PARP inhibitors or platinum-based chemotherapy. Cancer Res; 70(15); 6268-76. (C) 2010 AACR.
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| 10. |
- Issaeva, Natalia
(författare)
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Selective induction of apoptosis in tumors by small molecules reactivating p53
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Lesions in the p53 pathway that inactivate p53 tumor suppression function occur with extremely high frequency in cancer, targeting almost all tumors regardless of patient age or tumor type. This study is aimed at developing approaches for the selective elimination of tumors carrying defects in the p53 pathway, namely protection of p53 from degradation by its own destructor HDM2 in wild type p53-carrying tumors and refolding of p53 by small molecules in mutant p53-containing tumors. Among genes known to be involved in tumor development, the p53 gene has an exclusive status. Mutations of p53 gene occur in around 50% of human tumors. The great majority of p53 mutaitons are point missense mutations resulting in a substitution of only one amino acid residue that causes unfolding of the DNA binding domain. These particular features of p53 alterations in tumors, taken together with the observation that mutant p53 proteins accumulate at high levels in tumor cells, makes the strategy aimed at reactivation of p53 quite feasible. We identified and characterized a small molecule, PRIMA-1, which restored the sequencespecific DNA binding and active conformation to mutant p53 proteins. In vivo studies in mice revealed an antitumor effect with no apparent toxicity. We also showed that rationally designed nine-residue peptide CDB3 that bound to the tumor suppressor p53 and stabilized it against denaturation in vitro, binds p53 in cells, restores the active conformation and transcription transactivation function to mutant p53. These molecules,PRIMA-1 and CDB3, may serve as a lead compounds for the development of anticancer drugs targeting mutant p53. In wt p53-carrying human tumors, p53 pathway is disrupted via several mechanisms, including overexpression of p53's negative regulator, HDM2, or loss of expression of p14ARF via deletion of INK4a/ARF locus or methylation of p14ARF promoter. Given the central role of HDM2/ p 14ARF in control of p53 function, the potential for non-genotoxic cancer therapies based on the inhibition of HDM2 in tumors expressing wt p53 is of considerable interest. Restoration of p53 function by blocking p53/HDM2 interaction would result in induction of apoptosis in tumors while inducing transient growth arrest in normal cells, as it was shown that normal cells are less sensitive to p53 activation. This provides a powerful new approach to target tumor cells selectively. We have screened the library of low molecular weight compounds for molecules that can suppress the growth of human tumor cells in a wtp53-dependent manner, and identified the compound, named RITA, that binds p53 protein, prevents p53/HDM2 interaction in vitro and in vivo and affects p53's interaction with several negative regulators. RITA induces expression of p53 target genes and massive apoptosis in various tumor cells lines carrying wtp53. RITA demonstrates substantial p53-dependent anti-tumor effect in vivo. The strong anti-tumor activity of RITA exceeds the standard criteria of National Cancer Institute for candidate drugs.
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