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  • Open access
  • 125 Reads
LINP1 lncRNA expression profile is modulated in response to DNA damage

Rapidly dividing cancer cells show elevated levels of DNA double-strand breaks (DSBs) resulting from replication stress and linked to genome instability. To verify the hypothesis that a low level of endogenous replicative DNA damage may impact gene expression programs and cell biology features relevant to cancer progression, we used DNA ligase I (LigI) defective 46BR.1G1 fibroblasts, deriving from a patient who died at 19 for lymphoma, and the 7A3 cell clone, obtained from 46BR.1G1 by stably expressing ectopic wild-type LigI. LigI deficiency impairs maturation of newly synthesized DNA and increases the number of DSBs and γH2AX foci, features associated with genome instability commonly found also in pre-neoplastic lesions. In order to decipher the strategy used to cope with replicative DNA damage, we have compared gene expression profiles in 46BR.1G1 and 7A3 cells. Among the differentially expressed genes, we identified a group of long noncoding RNAs (lncRNAs) which show significant transcriptional alteration in 46BR.1G1 cells, and appear to be relevant for cancer progression. An interesting up-regulated lncRNA in 46BR.1G1 cells is LINP1 (lncRNA in nonhomologous end joining (NHEJ) pathway 1) which has been shown to be involved in DNA repair. We have observed that LINP1 up-regulation contributes to proliferation and survival of 46BR.1G1 that could account for genome instability. Moreover, we observed that LINP1 is upregulated at later times in control human fibroblasts exposed to exogenous sources of DNA damage. Our observations support the notion that LINP1 lncRNA targeting could reduce the DNA repair efficacy of tumour cells.

* These authors contributed equally to this work.

  • Open access
  • 119 Reads
Space and time in the universe of the cell nucleus after ionizing radiation attacks: a comparison of cancer and non-cancer cell response

Application of ionizing radiation has an increasing impact on bio-medical research, and cancer diagnosis and treatment. Nevertheless, there are a lot of open questions concerning the understanding of radiation DNA damaging mechanisms and repair processes within the light of radio-sensitivity and thus, individualized medical applications. The three-dimensional architecture of genomes on the micro-, meso- and nano-scale acts in combination with epigenetic modifications as an important player of gene regulation and, consequently, fundamental biological processes such as DNA damage response and repair. So far only little is known about the impact of chromatin architecture on DNA double strand break (DSB) repair pathway selection and progression at individual damage sites. How does a cell nucleus manage DSBs and re-organize the chromatin towards functionally intact repair units? Is there a radiosensitivity-related difference in this reaction? We present investigations of spatial and topological parameters of chromatin and repair foci during a time period of repair to glimpse key aspects related to these questions. Nano-probing of radiation-induced chromatin damage sites and the recruited DNA repair proteins in combination with super-resolution Single Molecule Localization Microscopy (SMLM) are powerful methods for geometric and topological analyses of these structures in single cells and single DSB sites and, thus, to study mechanisms of their formation and repair pathway regulation. We used variable tools for such investigations based on image-free high-precision SMLM, nano-scaled molecule distribution analyses, appropriate metrics following Ripley´s distance frequencies and cluster formation analyses, as well as topological quantifications employing persistence homology. Comparing the topology of repair foci by persistence homology suggests general similarities in repair cluster formation, indicating a well-defined non-random, molecule topology at given time points during repair. However, at the same time, the data reveal a specific nano-architecture of DNA damage foci depending on the chromatin domain and cell type. Showing how chromatin architecture around complex damage sites and repair focus nano-architecture may contribute to ongoing repair process, our studies contribute to the molecular understanding of cellular radiation response and its regulation in cancer and non-cancer cells at sub-light microscopic chromatin levels.

  • Open access
  • 80 Reads
The cytotoxic effect of 64Cu/NOTA-terpyridine platinum conjugate, as a novel chemoradiotherapy agent

Colorectal cancer is one of the most prevalent cancers worldwide that displays both intrinsic and acquired resistance to platinum-based chemotherapeutic agents (Pt-CAs). To overcome such resistance, new classes of Pt-CAs have been proposed, including terpyridine (TP) compounds that targets the G-quadruplex tertiary structure of DNA. Additionally, recent studies indicate a maximum chemoradiation benefit, when radiation is administered with Pt-CAs at their highest concentrations in cancer cell DNA. Accordingly, we synthesized a novel chemoradiotheranostic agent by conjugating a TP moiety with 64Cu (64Cu-NOTA-TP). The in-vitro cytotoxic effects, cellular uptake, internalization and efflux of 64Cu-NOTA-TP was measured for a colorectal cancer (HCT116) and normal fibroblast (GM05757) cells. Radiolabelling NOTA-TP with 64Cu resulted in 17530-, 40083- and 66000-fold enhancements in its cytotoxicity against HCT116 cells (EC50=0.017±0.004, 0.012±0.006 and 0.005±0.0002µM) as compared to coldCu-NOTA-terpyridine (EC50 = 298 ± 2, 481 ± 25 and 330 ± 51µM) at 24, 48 and 72h post-administration, respectively. More importantly, the cytotoxicity of the 64Cu-conjugate toward the HCT116 cells was about 3.8-fold higher than that of GM05757 cells at 24 and 72h. This result was consistent with a 2-3-fold higher internalization of 64Cu-conjugate in HCT116 cells relative to GM05757 cells at similar times. The internalized activity of the 64Cu-conjugate steadily increased from 0.04 ± 0.02% to 18.7±2.8% over 24h incubation time. Moreover, efflux kinetics of the 64Cu-conjugate showed that more than 40% of internalized activity was retained by cancer cells over a 24h. In conclusion, this work presents a novel chemoradiotherapeutic agent with considerable potential for targeted cancer treatment combined with radioisotope imaging.

  • Open access
  • 121 Reads
Heterogeneity of biomarker expression in clinical urine biopsies

Bladder cancer (BC) often requires lifetime monitoring due to its high recurrence rate. Exfoliated bladder cancer cells (EBCCs) may express a series of different biomarkers according to its epithelial-mesenchymal transition (EMT) status, a phenomenon characterized by loss of intercellular adhesion, enhanced cell motility, and cancer invasion. Here, we demonstrated the clinical heterogeneity of EBCCs using an integrated microfluidic assay to separate various EMT subtypes of EBCCs in real-time and under high-throughput based on the principle of inertial focusing. Enriched cells from BC patient-derived urine bladder wash samples were isolated based on cell size and characterized by antibodies targeting EMT biomarkers such as cytokeratin (CK), vimentin (VIM), survivin, and epidermal growth factor receptor (EGFR). This rapid, non-invasive method demonstrates high efficiency of cancer cell recovery under the optimal flow rate and the specific retrieval of various EMT phenotype cell fractions from respective device outlets. The evaluation of clinical samples revealed a vast amount of tumor heterogeneity, reflecting different EMT phenotypes, which can correlate with drug resistance and tumor dormancy. Overall, the separation of heterogeneous clinical samples can better facilitate routine screening procedures and greatly enhance personalized treatment.

  • Open access
  • 91 Reads
Gamma Radiation Induces Apoptosis by Over Expression of PERP Effector of p53-dependent Cell Death in HeLa Cells.

Ionized radiation leads to a modulation of the expression of many genes. Identification of specific genes may allow the determination of pathways important in radiation responses. It was found that PERP induces apoptosis when it is overexpressed. However, the mechanism by which PERP induces apoptosis is still unknown. The objectives of this study were to determine the configuration of several genes in response to gamma radiation treatment. Novel treatment strategy to increase the cancer cell sensitivity to radiotherapy by modulation of the PERP expression could be developed for cervical cancers.

Cervical cancer cells were incubated for several periods after were exposed to various doses of gamma radiation, MTT assay were used to explore propagation of HeLa cells, apoptotic Index (AI) were measured using fluorescent microscopy by estimating apoptotic morphological features. While, signalling pathway analyses were performed on up-regulated genes which evaluated using microarray technology.

From the results of this study, the proliferation of HeLa cervical cancer cells exposed to gamma radiation was inhibited proportionally with dose and time after exposure. Also, apoptotic morphological features, such as shrinkage of the cell and formation of apoptotic bodies, was clearly visible under the microscope for irradiated HeLa cells. after 48 h. exposure to different doses of gamma radiation, the dose of 32 Gy was specified as an AI dose. The mRNA levels of pro-apoptotic genes such as, PERP; BAX; CASP9; TRAF3 and other factors detected by microarray after treatment with gamma radiation were up-regulated. Whereas, many anti-apoptosis factors were down-regulated. P53 pathway were significantly reinforced after pathway analysis for up- and dawn- regulated genes. For conclusion, gamma radiation induces apoptosis by over expression of PERP factor and p53-dependent cell death in cervical carcinoma HeLa cells.

  • Open access
  • 193 Reads
Interplay between cellular metabolism and the DNA damage response

Metabolism is a fundamental cellular mechanism. Its regulation is crucial to maintain cellular homeostasis in response to fluctuation in energy demands and in the availability of nutrients. However, metabolic reactions can be harmful for cells, for example by leading to an increase in oxidative stress or through the generation of toxic by-products, which can in turn damage DNA. To deal with these insults, cells have evolved sophisticated DNA damage response (DDR) pathways that allow for the maintenance of genome integrity. Recent years have seen remarkable progress in unraveling the diverse mechanisms of the DDR. Through such work, it has also emerged that cellular metabolic regulation not only generates DNA damage but also impacts on DNA repair, yet a systematic analysis aimed at identifying interactions between metabolic alterations and the DDR remains unreported. Here, we have functionally explored such interplay, by taking a global and unbiased genetic approach.

By utilizing a pooled CRISPR-Cas9 sgRNA library targeting some 3,000 metabolic genes, we have determined the effects on DNA repair following the induction of DNA double-strand breaks induced by the chemotherapeutic etoposide, an inhibitor of topoisomerase 2. Through a phenotypic FACS-based screen with gH2AX used as a marker of DNA damage, we have identified metabolic genes that are required for DNA repair. Candidate genes have been prioritized and validated via an arrayed CRISPR screen based on high-throughput microscopy measuring multiple parameters, across different cell lines and DNA double-strand break inducing agents. We will present data on how metabolic genes impact on DNA repair.

Our research sheds light on how the fundamental cellular process of DNA damage maintenance is affected by alterations in cellular metabolism. Moreover, understanding the dynamic interplay between metabolic factors and DDR is also of crucial importance to better design efficient cancer treatments, since cancers are both genetic and metabolic diseases.

  • Open access
  • 56 Reads
The pluripotency transcription factor Oct4 contributes to head and neck squamous cell carcinoma radioresistance via regulation of DNA repair and the stem cell phenotype

Despite being the sixth most common cancer type worldwide, head and neck squamous cell carcinoma (HNSCC) exhibits low five-year survival rates for advanced-stage patients. The local control probability after radiotherapy crucially depends on the eradication of cancer stem cells (CSCs). This pluripotent sub-population of tumor cells is characterized by an active DNA repair and, consequently, an enhanced radio(chemo)therapy resistance. This study provides evidence that the CSC-related transcription factor Oct4 contributes to HNSCC radioresistance by regulating the DNA damage response and stem cell phenotype.

In a siRNA-mediated Oct4 knockdown model, we observed reduced self-renewal capacity and partial radiosensitization of HNSCC cell lines accompanied by decreased expression of the cell cycle checkpoint kinases Chk-1 and WEE1. Consequently, Oct4 knockdown impaired the G2 checkpoint induction after irradiation, linking Oct4 to the HNSCC DNA damage response. Upon CRISPR/Cas9-mediated knockout of the pluripotency-related isoform Oct4 A, radiosensitization of HNSCC cells could only be achieved in combination treatment with the PARP inhibitor Olaparib. In addition, irradiation-induced up-regulation of DNA repair genes, like the homologous recombination repair (HRR) gene BRCA1, was abolished in Oct4 A knockout cells, indicating that Oct4 A depletion leads to HRR deficiency in HNSCC cells.

Further analysis of the Oct4-correlating gene signature in the HNSCC TCGA patient dataset identified the HRR genes PSMC3IP and RAD54L showing a significant correlation with the overall survival of radiotherapy-treated HNSCC patients. siRNA-mediated knockdown of PSMC3IP and RAD54L reduced the HNSCC self-renewal capacity and clonogenic cell survival after irradiation, emphasizing the interplay between DNA repair and the CSC phenotype in HNSCC radioresistance mechanisms.

All in all, the involvement of Oct4 in the regulation of DNA repair and cell cycle progression provides new insights into HNSCC radioresistance and opens possibilities for combination therapy with PARP inhibitors.

  • Open access
  • 116 Reads
Determination of the effect of selected low fluoride concentrations on migratory abilities of human glioma U-87MG cell line.

Fluorine (F) is an element that belongs to the group of halogens. Small amounts of fluoride are necessary for the proper development of bones and teeth. However, increased intake of fluorine and continuous exposure has negative effects on the human organism. Some recent works have shown that fluoride affects many metabolic pathways that can theoretically be involved in the development of invasive potential in many types of cancers, including brain neoplasms. In light of recent studies, the influence of fluoride on the invasiveness of cancer cells seems highly probable but is practically unexplored.

„Wound healing” assay
After 72 hours or three months of passaging in appropriate NaF concentrations (0.1-10 µM), U-87MG cells were grown in 6-well plates (controls + NaF concentrations indicated). After reaching confluence (~ 80%), the cell layers were scratched with 200 µl pipette tips and washed with PBS to remove cell debris. Fresh medium without serum was added to each well and the wound closure was visualized at 0, 3, 6, 12, and 24 hours using a microscope.
Cell migration test
After 72 hours or three months of passage in appropriate NaF concentrations (0.1-10 µM), a total of 1 × 105 U-87MG cells (controls + 0.1-10 µM concentration of NaF, in serum-free EMEM containing 1% serum albumin bovine species ) were inoculated in the upper chamber of a 24-well Transwell system with a pore size of 8.0 µm. EMEM containing 10% FBS was added to the lower chamber. After incubation, non-migrating and non-invasive cells on the upper surface were removed with a cotton swab and cells on the lower surface were fixed with 4% paraformaldehyde and stained with Giemsa. Photographs were taken and cells were counted under the microscope.

Our observations showed that both in the case of short-term and long-term culture in the presence of sodium fluoride, the mobility of glioblastoma cells significantly increased. Importantly, the effect was visible at the lowest concentration (0.1 µM ) and increased at higher concentrations (1-10 µM) of NaF.

The results of these studies can shed new light on the therapeutic approach in people with brain tumors and draw attention to environmental factors such as fluoride, which may already hamper the treatment of patients at low doses. Considering the numerous processes taking place in the brain under the influence of fluoride, it seems extremely important to investigate the influence of this environmental toxin on the progression and development of brain tumors.

  • Open access
  • 123 Reads
Exploring sensitivity to replicative stress in BRCA deficient Triple Negative Breast Cancer

In Triple Negative Breast Cancer (TNBC), chemotherapy is the only systemic treatment and sustained remissions are rare. We propose to widen therapeutic options. About 30% TNBC tumors are BRCA1 deficient, presentoing defective DNA repair and increased sensitivity to genotoxic drugs. We hypothesized that BRCA-deficient TNBC are highly sensitive to replication stress inducing drugs, thus, opening new therapeutic perspectives. Our preliminary results shown that BRCA1-deficient TNBC cell lines and a CRISPR/Cas9 BRCA1 KO isogenic model display increased sensitivity to gemcitabine. Cell cycle distribution of gemcitabine treated BRCA1-deficient cells were characterized by an elevated Sub-G1 fraction caused by increased numbers of cells in replication catastrophe. This was illustrated by 80% of BRCA1-deficient cells showing persistent (48-72h post treatment) gH2AX staining in absence of RPA32 co-staining, whereas in the isogenic BRCA1 WT model gH2AX and RPA32 positive cell numbers started decreasing at 24h. Interestingly, we noted that in addition to replication catastrophe, BRCA-deficient cells treated with gemcitabine underwent aberrant mitosis as shown by a clear increase of micro-nuclei.

Interestingly, in vivo experiments appear to reproduce in vitro data. Indeed, a BRCA hyper methylated TNBC PDX, showed a higher sensitivity to gemcitabine than the BRCA1 WT. In conclusion, our data suggest that BRCA-deficient tumors are more sensitive to the replication poison Gemcitabine. Furthermore, this sensitivity seems to be mediated by an accentuated replicative stress response that is not well managed. Upon gemcitabine treatment, the cells undergo important DNA damage that leads to stalled replication forks, and DNA breakage. In the absence of BRCA1, the HR pathway is compromised, which leads to fork collapse and accumulation of single stranded DNA, therefore exhausting the pool of RPA within the cell and inducing Replicative catastrophe. In addition to deficient replication gemcitabine treated BRCA-deficient, but not BRCA-proficient cells, are subjected to mitotic catastrophe.

  • Open access
  • 147 Reads
NEIL3-mediated mitotic base excision repair of oxidative lesions at telomeres prevents senescence in hepatocellular carcinoma

While the repair of DNA double-strand breaks is known to be confined to different phases of the cell cycle and differentially activated at telomeres, less is known of compartmentalisation of base excision repair (BER). Here, we report that Endonuclease VIII like protein 3 (NEIL3) relocates to telomeres following oxidative DNA damage specifically during mitosis and recruits the APE1 to damaged telomeres. Using META-FISH, we demonstrate that NEIL3, but not NEIL1 or NEIL2, is required to initiate base excision repair at oxidised telomeres in mitotic cells, a process dependent on APE1 and Polβ. Repetitive exposure of oxidizing damage in NEIL3 depleted cells induced chromatin bridges and damaged telomeres. Interestingly, we identify that NEIL3 is elevated in Hepatocellular carcinoma (HCC), the most common type of a liver cancer, which correlates with poor survival. We demonstrate that HCC cell lines (6/6) depend on NEIL3 catalytic activity for survival and prevention of senescence, which is not the case for non-transformed cells where NEIL3 is dispensable. In conclusion, we demonstrate a novel function for NEIL3 in repair of oxidative DNA damage at telomeres in mitosis which is important to prevent senescence of HCC. Furthermore, these data suggest NEIL3 could be a target for therapeutic intervention of HCC.