TOPBP1 regulates RAD51 phosphorylation and chromatin loading and determines PARP inhibitor sensitivity

Topoisomerase IIβ-binding protein 1 (TOPBP1) participates in DNA replication and DNA damage response; however, its role in DNA repair and relevance for human cancer remain unclear. Here, through an unbiased small interfering RNA screen, we identified and validated TOPBP1 as a novel determinant whose loss sensitized human cells to olaparib, an inhibitor of poly(ADP-ribose) polymerase. We show that TOPBP1 acts in homologous recombination (HR) repair, impacts olaparib response, and exhibits aberrant patterns in subsets of human ovarian carcinomas. TOPBP1 depletion abrogated RAD51 loading to chromatin and formation of RAD51 foci, but without affecting the upstream HR steps of DNA end resection and RPA loading. Furthermore, TOPBP1 BRCT domains 7/8 are essential for RAD51 foci formation. Mechanistically, TOPBP1 physically binds PLK1 and promotes PLK1 kinase–mediated phosphorylation of RAD51 at serine 14, a modification required for RAD51 recruitment to chromatin. Overall, our results provide mechanistic insights into TOPBP1’s role in HR, with potential clinical implications for cancer treatment

Curcumin - from traditional medicine to the clinic

Curcumin (diferuloylmethane), a polyphenolic compound derived from turmeric, known as a spice and food-coloring agent, has been used for centuries to treat various illnesses. For the last few decades, extensive work has been done to establish the biological activities and pharmacological actions of curcumin. Curcumin possesses diverse pharmacological activities including anti-inflammatory, antioxidant, antiproliferative, pro-apoptotic and antiangiogenic. It is a well-known chemopreventive agent with potent anticarcinogenic activity in a wide variety of tumor cells. Moreover, it is known for antiarthritic and neuroprotective properties with a big potential role in the treatment of Alzheimer's disease. Curcumin has an outstanding safety profile and its lack of toxicity has been documented in the Phase I and II clinical trials. Although curcumin is poorly absorbed after ingestion and its low systemic bioavailability seems to limit the potential effects, multiple studies have documented that even low levels of physiologically achievable concentrations of curcumin may be sufficient for its chemopreventive and therapeutic activity against various human diseases. Recently, numerous approaches have been undertaken to improve the bioavailability of curcumin. This review summarizes the pleiotropic effects of curcumin and describes the recently identified molecular targets of curcumin

Comparative Study on Enzyme Immobilization Using Natural Hydrogel Matrices—Experimental Studies Supported by Molecular Models Analysis

Currently, great attention is focused on conducting manufacture processes using clean and eco-friendly technologies. This research trend also relates to the production of immobilized biocatalysts of industrial importance using matrices and methods that fulfill specified operational and environmental requirements. For that reason, hydrogels of natural origin and the entrapment method become increasingly popular in terms of enzyme immobilization. The presented work is the comparative research on invertase immobilization using two natural hydrogel matrices—alginate and gelatin. During the study, we provided the molecular insight into the structural characteristics of both materials regarding their applicability as effective enzyme carriers. In order to confirm our predictions of using these hydrogels for invertase immobilization, we performed the typical experimental studies. In this case, the appropriate conditions of enzyme entrapment were selected for both types of carrier. Next, the characterization of received invertase preparations was made. As a final experimental result, the gelatin-based hydrogel was selected as an effective carrier for invertase immobilization. Hereby, using mild conditions and a pro-ecological, biodegradable matrix, it was possible to obtain very stable and reactive biocatalyst. The choice of gelatin-immobilized invertase preparation was compatible with our predictions based on the molecular models of hydrogel matrices and enzyme used

Nuclear and mitochondrial genome responses in HeLa cells treated with inhibitors of mitochondrial DNA expression

The influence of mutations in the mitochondrial DNA (mtDNA) on the bioenergetic metabolism of the cell is still poorly understood. Many of the mutations in the mtDNA affect the expression of the mitochondrial genome. Investigations on cells from patients are not easy, especially as the mitochondrial DNA is heteroplasmic and this state is changed in culture. Moreover, the nuclear background and the mitochondrial haplotype may affect the behaviour of cells. Transfer of patient mitochondria to rho zero cell lines is also not optimal as these cells in general have many nuclear changes which may also affect cell behaviour. Thus, we decided to use inhibitors of mitochondrial genome expression, such as thiamphenicol, ethidium bromide and dideoxycytidine to investigate the bioenergetic metabolism of HeLa cells. We found that oxidative phosphorylation and glycolysis participate equally in ATP production in HeLa cells and that decreased activity of the respiratory chain leads to increased glycolysis and the reduction of cell growth. Insufficient ATP production in the oxidative phosphorylation process was not compensated by increased proliferation of the mitochondria. However, we were able to show that there are some mechanisms compensating limited expression of the mitochondrial genome within the mitochondria. Experiments with dideoxycytidine revealed that 10-fold decrease of the mtDNA copy number resulted in almost normal activity of cytochrome c oxidase. We found that mtDNA depletion is compensated mostly on the level of RNA metabolism in the mitochondria. Thus, our results are in agreement with the hypothesis that transcription initiation rather than mtDNA copy number is a rate limiting factor for expression of the mitochondrial genome

Increased acetylation of lysine 317/320 of p53 caused by BCR-ABL protects from cytoplasmic translocation of p53 and mitochondria-dependent apoptosis in response to DNA damage

Chronic myeloid leukemia (CML) is a disorder of hematopoietic stem cells caused by the expression of BCR-ABL. Loss of p53 has not been implicated as important for the development of CML. Mutations in p53 protein are infrequent, however they correlate with the disease progression. The absence of p53 mutations does not exclude the possibility that other dysfunctions play an important role in CML pathology. Acetylation represents a very potent posttranslational mechanism regulating p53 stability, transcriptional activity and localization. In this study we have investigated whether the expression of BCR-ABL could influence the acetylation of p53, specifically at lysine 317/320 (K317/K320), which has been shown to regulate nuclear export and transcription-independent apoptotic activity of p53. We found that BCR-ABL expression increases K317 acetylation of p53 and is able to prevent a drop in acetylation observed upon DNA damage, followed by translocation of p53 to the cytoplasm and by Bax activation. We have shown that this site plays a crucial role in the regulation of p53 localization and p53-dependent, Bax-mediated apoptosis. Our study presents a novel BCR-ABL-dependent mechanism protecting from DNA-damage-induced cell death. It can, in addition to already known mechanisms, explain the resistance to p53-dependent apoptosis observed in CML cells expressing wt p53. We propose that the acetyltransferases regulating the p53 acetylation could be an interesting and potent target for therapeutic intervention

Increased acetylation of lysine 317/320 of p53 caused by BCR-ABL protects from cytoplasmic translocation of p53 and mitochondria-dependent apoptosis in response to DNA damage

Chronic myeloid leukemia (CML) is a disorder of hematopoietic stem cells caused by the expression of BCR-ABL. Loss of p53 has not been implicated as important for the development of CML. Mutations in p53 protein are infrequent, however they correlate with the disease progression. The absence of p53 mutations does not exclude the possibility that other dysfunctions play an important role in CML pathology. Acetylation represents a very potent posttranslational mechanism regulating p53 stability, transcriptional activity and localization. In this study we have investigated whether the expression of BCR-ABL could influence the acetylation of p53, specifically at lysine 317/320 (K317/K320), which has been shown to regulate nuclear export and transcription-independent apoptotic activity of p53. We found that BCR-ABL expression increases K317 acetylation of p53 and is able to prevent a drop in acetylation observed upon DNA damage, followed by translocation of p53 to the cytoplasm and by Bax activation. We have shown that this site plays a crucial role in the regulation of p53 localization and p53-dependent, Bax-mediated apoptosis. Our study presents a novel BCR-ABL-dependent mechanism protecting from DNA-damage-induced cell death. It can, in addition to already known mechanisms, explain the resistance to p53-dependent apoptosis observed in CML cells expressing wt p53. We propose that the acetyltransferases regulating the p53 acetylation could be an interesting and potent target for therapeutic intervention

Downregulation of BRCA1 protein in BCR-ABL1 leukemia cells depends on stress-triggered TIAR-mediated suppression of translation

<div><p>BRCA1 tumor suppressor regulates crucial cellular processes involved in DNA damage repair and cell cycle control. We showed that expression of BCR-ABL1 correlates with decreased level of BRCA1 protein, which promoted aberrant mitoses and aneuploidy as well as altered DNA damage response. Using polysome profiling and luciferase-BRCA1 3’UTR reporter system here we demonstrate that downregulation of BRCA1 protein in CML is caused by inhibition of BRCA1 mRNA translation, but not by increased protein degradation or reduction of mRNA level and half-life. We investigated 2 mRNA-binding proteins – HuR and TIAR showing specificity to AU-Rich Element (ARE) sites in 3’UTR of mRNA. BCR-ABL1 promoted cytosolic localization of TIAR and HuR, their binding to BRCA1 mRNA and formation of the TIAR-HuR complex. HuR protein positively regulated BRCA1 mRNA stability and translation, conversely TIAR negatively regulated BRCA1 translation and was found localized predominantly in the cytosolic stress granules in CML cells. TIAR-dependent downregulation of BRCA1 protein level was a result of ER stress, which is activated in BCR-ABL1 expressing cells, as we previously shown. Silencing of TIAR in CML cells strongly elevated BRCA1 level. Altogether, we determined that TIAR-mediated repression of BRCA1 mRNA translation is responsible for downregulation of BRCA1 protein level in BCR-ABL1 –positive leukemia cells. This mechanism may contribute to genomic instability and provide justification for targeting PARP1 and/or RAD52 to induce synthetic lethality in “BRCAness” CML and BCR-ABL1 –positive ALL cells.</p></div