Vrije Universiteit Amsterdam
Medical Oncology
Present cancer treatment strategies are based on the assumption that a therapy may work (“response”) or not work (“no-response”). However, the existing evidence suggests that current cancer treatment modalities may also have a... more
Present cancer treatment strategies are based on the assumption that a therapy may work (“response”) or not work (“no-response”). However, the existing evidence suggests that current cancer treatment modalities may also have a cancer-promoting effect in part of the patients. In this paper, some relevant data are reviewed suggesting that surgery, irradiation, chemotherapy and immunotherapy can stimulate tumor growth / metastatic spread and decrease survival of patients in certain subgroups. Thus, results of cancer treatment may be improved by detection and use of biomarkers that correlate with positive or negative therapeutic effects. Small trials based on groups with differing biomarkers rather than large phase III trials may aid the development and efficacy testing of new anticancer drugs. Moreover, ignoring biomarkers that correlate with positive or negative therapeutic effect may not be compatible anymore with the ethical principle “First Do No Harm”.
- by john jacobs and +1
- •
- Surgery, Cancer, Chemotherapy, Cancer treatment
Deoxycytidine kinase (dCK) is essential for phosphorylation of natural deoxynucleosides and analogs, such as gemcitabine and cytarabine, two widely used anticancer compounds. Regulation of dCK is complex, including Ser-74 phosphorylation.... more
Deoxycytidine kinase (dCK) is essential for phosphorylation of natural deoxynucleosides and analogs, such as gemcitabine and cytarabine, two widely used anticancer compounds. Regulation of dCK is complex, including Ser-74 phosphorylation. We hypothesized that dCK could be regulated by two additional mechanisms: micro-RNA (miRNA) and promoter methylation. Methylation-specific PCR (MSP) revealed methylation of the 3 ′ GC box in three out of six cancer cell lines. The 3 ′ GC box is located at the dCK promoter region. The methylation status was related to dCK mRNA expression. TargetScan and miRanda prediction algorithms revealed several possible miRNAs targeting dCK and identified miR-330 (micro-RNA 330) as the one conserved between the human, the chimpanzee, and the rhesus monkey genomes. Expression of miR-330 in various colon and lung cancer cell lines, as measured by QRT-PCR, varied five-fold between samples and correlated with in-vitro gemcitabine resistance (R = 0.82, p = 0.04). Exposure to gemcitabine also appeared to influence miR-330 levels in these cell lines. Furthermore, in our cell line panel, miR-330 expression negatively correlated with dCK mRNA expression (R = 0.74), suggesting a role of miR-330 in post-transcriptional regulation of dCK. In conclusion, the 3 ′ GC box and miR-330 may regulate dCK expression in cancer cells.
- by Jasmina Hodzic and +1
- •
- Genetics, Neuroscience, Oncology, Biotechnology
Present cancer treatment strategies are based on the assumption that a therapy may work (“response”) or not work (“no-response”). However, the existing evidence suggests that current cancer treatment modalities may also have a... more
Present cancer treatment strategies are based on the assumption that a therapy may work (“response”) or not work (“no-response”). However, the existing evidence suggests that current cancer treatment modalities may also have a cancer-promoting effect in part of the patients. In this paper, some relevant data are reviewed suggesting that surgery, irradiation, chemotherapy and immunotherapy can stimulate tumor growth / metastatic spread and decrease survival of patients in certain subgroups. Thus, results of cancer treatment may be improved by detection and use of biomarkers that correlate with positive or negative therapeutic effects. Small trials based on groups with differing biomarkers rather than large phase III trials may aid the development and efficacy testing of new anticancer drugs. Moreover, ignoring biomarkers that correlate with positive or negative therapeutic effect may not be compatible anymore with the ethical principle “First Do No Harm”.
- by Jasmina Hodzic and +1
- •
- Cancer, Chemotherapy, Medical Oncology, Cancer radiotherapy
The centrosome plays a key role in cancer invasion and metastasis. However, it is unclear how abnormal centrosome numbers are regulated when prostate cancer (PCa) cells become metastatic. CP110 was previously described for its... more
The centrosome plays a key role in cancer invasion and metastasis. However, it is unclear how abnormal centrosome numbers are regulated when prostate cancer (PCa) cells become metastatic. CP110 was previously described for its contribution of centrosome amplification (CA) and early development of aggressive cell behaviour. However its regulation in metastatic cells remains unclear. Here we identified miR-129-3p as a novel metastatic microRNA. CP110 was identified as its target protein. In PCa cells that have metastatic capacity, CP110 expression was repressed by miR-129-3p. High miR-129-3p expression levels increased cell invasion, while increasing CP110 levels decreased cell invasion. Overexpression of CP110 in metastatic PCa cells resulted in a decrease in the number of metastasis. In tissues of PCa patients, low CP110 and high miR-129-3p expression levels correlated with metastasis, but not with the expression of genes related to EMT. Furthermore, overexpression of CP110 in metastatic PCa cells resulted in excessive-CA (E-CA), and a change in F-actin distribution which is in agreement with their reduced metastatic capacity. Our data demonstrate that miR-129-3p functions as a CA gatekeeper in metastatic PCa cells by maintaining pro-metastatic centrosome amplification (CA) and preventing anti-metastatic E-CA.
Background: Radiotherapy is one of the mainstays in the treatment for cancer, but its success can be limited due to inherent or acquired resistance. Mechanisms underlying radioresistance in various cancers are poorly understood and... more
Background: Radiotherapy is one of the mainstays in the treatment for cancer, but its success can be limited due to inherent or acquired resistance. Mechanisms underlying radioresistance in various cancers are poorly understood and available radiosensitizers have shown only modest clinical benefit. There is thus a need to identify new targets and drugs for more effective sensitization of cancer cells to irradiation. Compound and RNA interference high-throughput screening technologies allow comprehensive enterprises to identify new agents and targets for radiosensitization. However, the gold standard assay to investigate radiosensitivity of cancer cells in vitro, the colony formation assay (CFA), is unsuitable for high-throughput screening.
Lung cancer is the most common cancer worldwide and on top of that has a very poor prognosis, which is reflected by a 5-year survival rate of 5% to 15%. Radiotherapy is an integral part of most treatment regimens for this type of tumor,... more
Lung cancer is the most common cancer worldwide and on top of that has a very poor prognosis, which is reflected by a 5-year survival rate of 5% to 15%. Radiotherapy is an integral part of most treatment regimens for this type of tumor, often combined with radiosensitizing cytotoxic drugs. In this study, we identified many genes that could potentially be exploited for targeted radiosensitization using a genome-wide siRNA screen in non–small cell lung cancer (NSCLC) cells. The screen identified 433 siRNAs that potentially sensitize lung cancer cells to radiation. Validation experiments showed that knockdown of expression of Forkhead box M1 (FOXM1) or microtubule- associated serine/threonine kinase-like (MASTL) indeed causes radiosensitization in a panel of NSCLC cells. Strikingly, this effect was not observed in primary human fibroblasts, suggest-
ing that the observed radiosensitization is specific for cancer cells. Phosphoproteomics analyses with and without irradia- tion showed that a number of cell-cycle–related proteins were significantly less phosphorylated after MASTL knockdown in comparison to the control, while there were no changes in the levels of phosphorylation of DNA damage response proteins. Subsequent analyses showed that MASTL knockdown cells respond differently to radiation, with a significantly shortened G2–M phase arrest and defects in cytokinesis, which are fol- lowed by a cell-cycle arrest. In summary, we have identified many potential therapeutic targets that could be used for radiosensitization of NSCLC cells, with MASTL being a very promising and druggable target to combine with radiotherapy. Mol Cancer Ther; 14(6); 1434–44. !2015 AACR.
ing that the observed radiosensitization is specific for cancer cells. Phosphoproteomics analyses with and without irradia- tion showed that a number of cell-cycle–related proteins were significantly less phosphorylated after MASTL knockdown in comparison to the control, while there were no changes in the levels of phosphorylation of DNA damage response proteins. Subsequent analyses showed that MASTL knockdown cells respond differently to radiation, with a significantly shortened G2–M phase arrest and defects in cytokinesis, which are fol- lowed by a cell-cycle arrest. In summary, we have identified many potential therapeutic targets that could be used for radiosensitization of NSCLC cells, with MASTL being a very promising and druggable target to combine with radiotherapy. Mol Cancer Ther; 14(6); 1434–44. !2015 AACR.
Aims Endothelial cells (ECs) control vascular permeability by forming a monolayer that is sealed by extracellular junctions. Various mediators modulate the endothelial barrier by acting on junctional protein complexes and the therewith... more
Aims
Endothelial cells (ECs) control vascular permeability by forming a monolayer that is sealed by extracellular junctions. Various mediators modulate the endothelial barrier by acting on junctional protein complexes and the therewith con- nected F-actin cytoskeleton. Different Rho GTPases participate in this modulation, but their mechanisms are still partly resolved. Here, we aimed to elucidate whether the opening and closure of the endothelial barrier are asso-
ciated with distinct localized RhoA activities at the subcellular level.
Methods and results
Live fluorescence resonance energy transfer (FRET) microscopy revealed spatially distinct RhoA activities associated
with different aspects of the regulation of endothelial monolayer integrity. Unstimulated ECs were characterized by
hotspots of RhoA activity at their periphery. Thrombin receptor activation in the femoral vein of male wistar rats and
in cultured ECs enhanced RhoA activity at membrane protrusions, followed by a more sustained RhoA activity
associated with cytoplasmic F-actin filaments, where prolonged RhoA activity coincided with cellular contractility.
Unexpectedly, thrombin-induced peripheral RhoA hotspots were not spatially correlated to the formation of
large inter-endothelial gaps. Rather, spontaneous RhoA activity at membrane protrusions coincided with the
closure of inter-endothelial gaps. Electrical impedance measurements showed that RhoA signalling is essential for
this protrusive activity and maintenance of barrier restoration.
Conclusion
Spontaneous RhoA activity at membrane protrusions is spatially associated with closure, but not formation of inter- endothelial gaps, whereas RhoA activity at distant contractile filaments contributes to thrombin-induced disruption of junctional integrity. Thus, these data indicate that distinct RhoA activities are associated with disruption and re- annealing of endothelial junctions.
Keywords
Rho GTPase † FRET microscopy † Cytoskeleton † Endothelial function † Vasoactive agents
Endothelial cells (ECs) control vascular permeability by forming a monolayer that is sealed by extracellular junctions. Various mediators modulate the endothelial barrier by acting on junctional protein complexes and the therewith con- nected F-actin cytoskeleton. Different Rho GTPases participate in this modulation, but their mechanisms are still partly resolved. Here, we aimed to elucidate whether the opening and closure of the endothelial barrier are asso-
ciated with distinct localized RhoA activities at the subcellular level.
Methods and results
Live fluorescence resonance energy transfer (FRET) microscopy revealed spatially distinct RhoA activities associated
with different aspects of the regulation of endothelial monolayer integrity. Unstimulated ECs were characterized by
hotspots of RhoA activity at their periphery. Thrombin receptor activation in the femoral vein of male wistar rats and
in cultured ECs enhanced RhoA activity at membrane protrusions, followed by a more sustained RhoA activity
associated with cytoplasmic F-actin filaments, where prolonged RhoA activity coincided with cellular contractility.
Unexpectedly, thrombin-induced peripheral RhoA hotspots were not spatially correlated to the formation of
large inter-endothelial gaps. Rather, spontaneous RhoA activity at membrane protrusions coincided with the
closure of inter-endothelial gaps. Electrical impedance measurements showed that RhoA signalling is essential for
this protrusive activity and maintenance of barrier restoration.
Conclusion
Spontaneous RhoA activity at membrane protrusions is spatially associated with closure, but not formation of inter- endothelial gaps, whereas RhoA activity at distant contractile filaments contributes to thrombin-induced disruption of junctional integrity. Thus, these data indicate that distinct RhoA activities are associated with disruption and re- annealing of endothelial junctions.
Keywords
Rho GTPase † FRET microscopy † Cytoskeleton † Endothelial function † Vasoactive agents
Background: Radiotherapy is one of the mainstays in the treatment for cancer, but its success can be limited due to inherent or acquired resistance. Mechanisms underlying radioresistance in various cancers are poorly understood and... more
Background: Radiotherapy is one of the mainstays in the treatment for cancer, but its success can be limited due to inherent or acquired resistance. Mechanisms underlying radioresistance in various cancers are poorly understood and available radiosensitizers have shown only modest clinical benefit. There is thus a need to identify new targets and drugs for more effective sensitization of cancer cells to irradiation. Compound and RNA interference high-throughput screening technologies allow comprehensive enterprises to identify new agents and targets for radiosensitization. However, the gold standard assay to investigate radiosensitivity of cancer cells in vitro, the colony formation assay (CFA), is unsuitable for high-throughput screening.
Oncolytic adenoviruses represent a novel class of anticancer agents. Their efficacy in killing cancer cells is variable, suggesting that there is room for improvement. Host miRNAs have been shown to play important roles in susceptibility... more
Oncolytic adenoviruses represent a novel class of anticancer agents. Their efficacy in killing cancer cells is variable, suggesting that there is room for improvement. Host miRNAs have been shown to play important roles in susceptibility of cells to replication of different viruses. Here, we investigate if adenovirus replication in human prostate cancer cells is influenced by host cell
miRNA expression. To this end, we analyzed human miRNA expression in response to adenovirus infection and performed functional screens for lytic adenovirus replication using synthetic miRNA mimic and inhibitor libraries. Adenovirus infection generally reduced miRNA expression. On top of this nonspecific interference with miRNA biogenesis, a set of miRNAs including in particular miR-222 was found specifically reduced. Another set miRNAs was found to promote adenovirus-induced death of prostate cancer cells. In most cases, this did not stimulate adenovirus propagation. The exception was miR-26b. Overexpression of miR-26b inhibited adenovirus-induced NF-B activation, augmented adenovirus-mediated cell death, increased adenovirus progeny release and promoted adenovirus propagation and spread in several human prostate cancer cell lines. This suggests that miR-26b is particularly useful to be combined with oncolytic adenovirus for more effective treatment of prostate cancer.
miRNA expression. To this end, we analyzed human miRNA expression in response to adenovirus infection and performed functional screens for lytic adenovirus replication using synthetic miRNA mimic and inhibitor libraries. Adenovirus infection generally reduced miRNA expression. On top of this nonspecific interference with miRNA biogenesis, a set of miRNAs including in particular miR-222 was found specifically reduced. Another set miRNAs was found to promote adenovirus-induced death of prostate cancer cells. In most cases, this did not stimulate adenovirus propagation. The exception was miR-26b. Overexpression of miR-26b inhibited adenovirus-induced NF-B activation, augmented adenovirus-mediated cell death, increased adenovirus progeny release and promoted adenovirus propagation and spread in several human prostate cancer cell lines. This suggests that miR-26b is particularly useful to be combined with oncolytic adenovirus for more effective treatment of prostate cancer.
Reproducibility of hits from independent CRISPR or siRNA screens is poor. This is partly due to data normalization primarily addressing technical variability within independent screens, and not the technical differences between them. We... more
Reproducibility of hits from independent CRISPR or siRNA screens is poor. This is partly due to data normalization primarily addressing technical variability within independent screens, and not the technical differences between them. We present "rscreenorm", a method that standardizes the functional data ranges between screens using assay controls, and subsequently performs a piecewise-linear normalization to make data distributions across all screens comparable. In simulation studies, rscreenorm reduces false positives. Using two multiple-cell lines siRNA screens, rscreenorm increased reproducibility between 27 and 62% for hits, and up to 5-fold for non-hits. Using publicly available CRISPR-Cas screen data, application of commonly used median centering yields merely 34% of overlapping hits, in contrast with rscreenorm yielding 84% of overlapping hits. Furthermore, rscreenorm yielded at most 8% discordant results, whilst median-centering yielded as much as 55%. Rscreenorm ...
Deoxycytidine kinase (dCK) is essential for phosphorylation of natural deoxynucleosides and analogs, such as gemcitabine and cytarabine, two widely used anticancer compounds. Regulation of dCK is complex, including Ser-74 phosphorylation.... more
Deoxycytidine kinase (dCK) is essential for phosphorylation of natural deoxynucleosides and analogs, such as gemcitabine and cytarabine, two widely used anticancer compounds. Regulation of dCK is complex, including Ser-74 phosphorylation. We hypothesized that dCK could be regulated by two additional mechanisms: micro-RNA (miRNA) and promoter methylation. Methylation-specific PCR (MSP) revealed methylation of the 3 ′ GC box in three out of six cancer cell lines. The 3 ′ GC box is located at the dCK promoter region. The methylation status was related to dCK mRNA expression. TargetScan and miRanda prediction algorithms revealed several possible miRNAs targeting dCK and identified miR-330 (micro-RNA 330) as the one conserved between the human, the chimpanzee, and the rhesus monkey genomes. Expression of miR-330 in various colon and lung cancer cell lines, as measured by QRT-PCR, varied five-fold between samples and correlated with in-vitro gemcitabine resistance (R = 0.82, p = 0.04). Exposure to gemcitabine also appeared to influence miR-330 levels in these cell lines. Furthermore, in our cell line panel, miR-330 expression negatively correlated with dCK mRNA expression (R = 0.74), suggesting a role of miR-330 in post-transcriptional regulation of dCK. In conclusion, the 3 ′ GC box and miR-330 may regulate dCK expression in cancer cells.
- by Jasmina Hodzic and +1
- •
- Genetics, Neuroscience, Oncology, Biotechnology
The expression levels of constitutively active Akt, NF-úB and NF-úB-dependent antiapoptotic proteins in LNCaP, PC3, and DU145 prostate cancer cells were determined by Western blotting. Curcumin, Akt inhibitor SH-6, and siRNA-Akt were used... more
The expression levels of constitutively active Akt, NF-úB and NF-úB-dependent antiapoptotic proteins in LNCaP, PC3, and DU145 prostate cancer cells were determined by Western blotting. Curcumin, Akt inhibitor SH-6, and siRNA-Akt were used to sensitize cancer cells to TRAIL and to understand cross-talk between Akt and NF-úB signaling pathways and their role in resistance of prostate cancer cells to TRAIL-induced apoptosis. Results: Each cancer cell line studied expressed transcriptionally active NF-úB which was inhibited by curcumin at concentration range of 10 to 30 mM. LNCaP and PC3 cells but not DU145 cells expressed p-Akt kinase which was also inhibited by curcumin. Inhibition of the NF-úB and p-Akt by curcumin sensitized cancer cells to TRAIL-induced cytotoxicity. Since NF-úB is a downstream target of p-Akt, we investigated whether inhibition of NF-úB by curcumin is mediated through suppression of Akt activation. Treatment of PC3 cells with SH-6, a specific inhibitor of Akt, or transfection with siRNA-Akt, not only inhibited p-Akt but also abrogated the nuclear expression of NF-úB. Furthermore, treatment with SH-6 or selective inhibition of Akt through siRNA-Akt inhibited NF-úB and sensitized cells to TRAIL. In contrast, SH-6 failed to inhibit NF-úB or sensitize DU145 prostate cancer cells to TRAIL as these cells do not express p-Akt. Because expression of antiapoptotic Bcl-2, Bcl-xL and XIAP is regulated by NF-úB, both curcumin and SH-6 decreased the levels of these proteins in PC3 cells through inhibition of NF-úB. Further, gene silencing of Bcl-2 with siRNA-Bcl-2 sensitized PC3 cells to TRAIL. Conclusions: These data define a molecular pathway wherein curcumin sensitizes prostate cancer cells to TRAIL by inhibiting Akt-regulated NF-úB and NF-úB-dependent antiapoptotic Bcl-2, Bcl-xL and XIAP.
These authors equally contributed to the study references 1. Vogelzang NJ, Rusthoven JJ, Symanowski J et al. Phase III study of pemetrexed in combination with cisplatin versus cisplatin alone in patients with malignant pleural... more
These authors equally contributed to the study references 1. Vogelzang NJ, Rusthoven JJ, Symanowski J et al. Phase III study of pemetrexed in combination with cisplatin versus cisplatin alone in patients with malignant pleural mesothelioma. J Clin Oncol 2003; 21: 2636-2644. 2. Righi L, Capotti MG, Ceppi P et al. Thymidylate synthase but not excision repair cross-complementation group 1 tumor expression predicts outcome in patients with malignant pleural mesothelioma treated with pemetrexed-based chemotherapy. J Clin Oncol 2010; 28: 1534-1539. 3. Zucali P, Destro A, Ceresoli G et al. The role of thymidylate synthase (TS) and excision repair cross-complementing group 1 (ERCC1) immunohistochemical expression in malignant letters to the editor Annals of Oncology 1560 | letters to the editor
Background: Approximately 10% of unselected non-small-cell lung cancer (NSCLC) patients responded to the epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) treatment. However, resistance mechanisms are not well... more
Background: Approximately 10% of unselected non-small-cell lung cancer (NSCLC) patients responded to the epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) treatment. However, resistance mechanisms are not well understood. We evaluated several potential biological markers of intrinsic EGFR-TKIs-resistance in NSCLC.
Background: This multicenter study evaluated three candidate microRNAs (miRNAs) (miR-21, miR-155 and miR-101) as potential biomarkers in intraductal papillary mucinous neoplasms (IPMNs) of the pancreas.
- by Sara Caponi and +2
- •
- Multivariate Analysis, Aged, microRNAs, Neoplasm Invasiveness
Women are usually advised to interrupt breastfeeding during chemotherapy for concerns of serious side-effects to the infant. However, the passage of cytotoxic drugs in human milk has been poorly studied.
Background: The selection of patients according to key genetic characteristics may help to tailor chemotherapy and optimize the treatment in Castration-Resistant Prostate Cancer (CRPC) patients. Functional polymorphisms within the... more
Background: The selection of patients according to key genetic characteristics may help to tailor chemotherapy and optimize the treatment in Castration-Resistant Prostate Cancer (CRPC) patients. Functional polymorphisms within the cytochrome P450 1B1 (CYP1B1) gene have been associated with alterations in enzymatic expression and activity and may change sensitivity to the widely used docetaxel regimen. Methods: CYP1B1 genotyping was performed on blood samples of 60 CRPC patients treated with docetaxel, using TaqMan probes-based assays. Association between CYP1B1-142C>G (leading to the 48ArgGly transition), 4326C>G (432LeuVal), and 4390A>G (453AsnSer) polymorphisms and treatment response, progression-free-survival (PFS) and overall-survival (OS) was estimated using Pearson χ 2 test, Kaplan-Meier curves and Log-rank test.
Purpose Oxaliplatin eVect in the treatment of colorectal cancer is improved upon combination with thymidylate synthase (TS) inhibitors. Pemetrexed is polyglutamated by the folylpolyglutamate synthase (FPGS) and blocks folate metabolism... more
Purpose Oxaliplatin eVect in the treatment of colorectal cancer is improved upon combination with thymidylate synthase (TS) inhibitors. Pemetrexed is polyglutamated by the folylpolyglutamate synthase (FPGS) and blocks folate metabolism and DNA synthesis by inhibiting TS, dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyltransferase (GARFT). The present study evaluates the pharmacological interaction between oxaliplatin and pemetrexed in colorectal cancer cells. Methods Human HT29, WiDr, SW620 and LS174T cells were treated with oxaliplatin and pemetrexed. Drug interaction was studied using the combination index method, while cell cycle was investigated with Xow cytometry. The eVects of drugs on Akt phosphorylation and apoptosis were studied with ELISA and Xuorescence microscopy, respectively. RT-PCR analysis was performed to assess whether drugs modulated the expression of pemetrexed targets and of genes involved in DNA repair (ERCC1 and ERCC2). Finally, platinum-DNA adduct levels were detected by ultra-sensitive multi-collector inductively coupled plasma mass spectrometry (ICP-MS). Results A dose-dependent inhibition of cell growth was observed after drug exposure, while a synergistic interaction was detected preferentially with sequential combinations. Oxaliplatin enhanced cellular population in the S-phase. Drug combinations increased apoptotic indices with respect to single agents, and both drugs inhibited Akt phosphorylation. RT-PCR analysis showed a correlation between the FPGS/(TS £ DHFR £ GARFT) ratio and pemetrexed sensitivity, as well as a downregulation of ERCC1, ERCC2, TS, DHFR and GARFT after drug exposure. In addition, pretreatment with pemetrexed resulted in an increase of oxaliplatin-DNA adducts. Conclusion These data demonstrate that oxaliplatin and pemetrexed synergistically interact against colon cancer cells, through modulation of cell cycle, inhibition of Akt phosphorylation, induction of apoptosis and modulation of gene expression.
- by Gareth Veal and +1
- •
- Flow Cytometry, Fluorescence Microscopy, Cell Cycle, DNA repair