EMX2 inhibits clear cell renal cell carcinoma progress via modulating Akt/FOXO3a pathway.

Empty spiracles homeobox 2 (EMX2) is initially identified as a key transcription factor that plays an essential role in the regulation of neuronal development and some brain disorders. Recently, several studies emphasized that EMX2 could as a tumor suppressor, but its role in human clear cell renal cell carcinoma (ccRCC) remains unclear. In the present study, we investigated the role and underlying mechanism of EMX2 in the regulation of ccRCC progress. Our results demonstrated that EMX2 expression was markedly decreased in ccRCC tissues and cell lines, and low EMX2 expression predicted the poor prognosis of ccRCC patients. In addition, forced expression of EMX2 significantly inhibited the cell growth, migration, and invasion in vitro, as well as ccRCC tumor growth in nude mice, via, at least in part, regulating Akt/FOXO3a pathway. In detail, EMX2 could attenuate the phosphorylation levels of Akt and FOXO3a, and increase FOXO3a expression without affecting total Akt expression in vivo and in vitro. Meanwhile, shRNA-mediated knockdown of FOXO3a expression could obviously attenuate the effects of EMX2 on cell growth, migration, invasion, and tumor growth. Furthermore, EMX2 could significantly attenuate the interaction between Akt and FOXO3a. Taken together, our results demonstrated that EMX2 could inhibit ccRCC progress through, at least in part, modulating Akt/FOXO3a signaling pathway, thus representing a novel role and underlying mechanism of EMX2 in the regulation of ccRCC progress.

Histone deacetylase 6 suppression of renal tubular epithelial cell promotes interstitial mineral deposition via alpha-tubulin acetylation.

Randall's plaque (RP) is derived from interstitial mineral deposition and is highly prevalent in renal calcium oxalate (CaOx) stone disease, which is predictive of recurrence. This study shows that histone deacetylase 6 (HDAC6) levels are suppressed in renal tubular epithelial cells in RP samples, in kidney tissues of hyperoxaluria rats, and in hyper-oxalate-treated or mineralized cultured renal tubular epithelial (MDCK) cells in vitro. Mineral deposition in MDCK cells was exacerbated by HDAC6 inhibition but alleviated by HDAC6 overexpression. Surprisingly, the expression of some osteogenic-associated proteins, were not increased along with the increasing of mineral deposition, and result of single-cell RNA sequencing of renal papillae samples revealed that epithelial cells possess lower calcific activity, suggesting that osteogenic-transdifferentiation may not have actually occurred in tubular epithelial cells despite mineral deposition. The initial mineral depositions facilitated by HDAC6 inhibitor were localized in extracellular dome rather than inside the cells, moreover, suppression of HDAC6 significantly increased the calcium content of co-cultured renal interstitial fibroblasts (NRK49F) and enhanced mineral deposition of indirectly co-cultured NRK49F cells, suggesting that HDAC6 may influence trans-MDCK monolayer secretion of mineral. Further experiments revealed that this regulatory role was partially alpha-tubulinLys40 acetylation dependent. Collectively, these results suggest that hyper-oxalate exposure led to HDAC6 suppression in renal tubular epithelial cells, which may contribute to interstitial mineral deposition by promoting alpha-tubulinLys40 acetylation. Therapeutic agents that influence HDAC6 activity may be beneficial in preventing RP and CaOx stone formation.

LncRNA MAGI2-AS3 inhibites tumor progression by up-regulating STAM via interacting with miR-142-3p in clear cell renal cell carcinoma.

Revealing the role of non-coding RNAs (ncRNAs) in inducing dysregulated pathological responses to external signals may identify therapeutic targets for inhibiting the progression of clear cell renal cell carcinoma (ccRCC). Non-coding RNAs belong to a class of RNA molecules that do not encode proteins but possess diverse biological functions, playing essential roles in the occurrence and development of metastatic and proliferative tumors. To investigate the impact of the upstream interaction between miR-142-3p and lncRNA MAGI2-AS3 on the tumor-suppressive activity of the STAM gene, we firstly conducted bioinformatics analysis to predict the upstream miRNAs of STAM and the upstream lncRNAs of the miRNAs through online databases (miRanda, miRDB, TargetScan, LncBase v2), which were further validated by the starBasev2.0 database. Subsequently, multiple experimental techniques were employed to validate these findings, including RT-qPCR, Western blotting, measurement of cellular functional activity, and luciferase reporter assays. Through these experimental methods, we provided compelling evidence regarding the role of miR-142-3p and MAGI2-AS3 in regulating STAM gene expression and functionality, revealing their potential significance in tumor suppression. Our research demonstrates the importance of the MAGI2-AS3/miR-142-3p/STAM signaling pathway axis in ccRCC. MAGI2-AS3 competes for binding with miR-142-3p, resulting in upregulated STAM gene expression. This upregulation inhibits tumor proliferation and metastasis in ccRCC cells. Conversely, overexpression of miR-142-3p or silencing of MAGI2-AS3 promotes tumor behavior, while downregulation of miR-142-3p inhibits the development of ccRCC. Targeting the MAGI2-AS3/miR-142-3p/STAM axis holds promise as a therapeutic strategy for ccRCC treatment.

ß-Arrestin2 promotes docetaxel resistance of castration-resistant prostate cancer via promoting hnRNP A1-mediated PKM2 alternative splicing.

PURPOSE: To investigate the influence of ß-arrestin2 on the docetaxel resistance in castration-resistant prostate cancer (CRPC) and elucidate the underlying molecular mechanisms. METHODS: PC3 and DU145 cells with stable ß-arrestin2 overexpression and C4-2 cells with stable ß-arrestin2 knockdown, were constructed via using lentivirus and puromycin selection. MTT and colony formation assays were carried out to investigate the effect of ß-arrestin2 expression on the docetaxel resistance of CRPC cells. Glycolysis analysis was used to assess the glycolytic capacity modulated by ß-arrestin2. GO enrichment analysis, gene set enrichment analysis and Spearman correlation test were carried out to explore the potential biological function and mechanism via using public data from GEO and TCGA. The expressions of PKM2, Phospho-PKM2, Phospho-ERK1/2 and hnRNP A1 were detected by western blot. Functional blocking experiments were carried out to confirm the roles of PKM2 and hnRNP A1 in the regulation of ß-arrestin2's biological functions via silencing PKM2 or hnRNP A1 expression in cells with stable ß-arrestin2 overexpression. Finally, nude mice xenograft models were established to confirm the experimental results of cell experiments. RESULTS: ß-Arrestin2 significantly decreased the sensitivity of CRPC cells to docetaxel stimulation, through enhancing the phosphorylation and expression of PKM2. Additionally, ß-arrestin2 increased PKM2 phosphorylation via the ERK1/2 signaling pathway and induced PKM2 expression in a post-transcriptional manner through an hnRNP A1-dependent PKM alternative splicing mechanism, rather than by inhibiting its ubiquitination degradation. CONCLUSION: Our findings indicate that the ß-arrestin2/hnRNP A1/PKM2 pathway could be a promising target for treating docetaxel-resistant CRPC.

Interleukins-6 -174G/C (rs1800795) and -572C/G (rs1800796) polymorphisms and prostate cancer risk.

Prostate cancer (PCa) is an aggressive cancer influenced by a complex interplay of genetic and environmental factors. Amongst these risk factors, the impact of Interleukin6 (IL6) gene polymorphisms in PCa risk has received a lot of attention. IL-6 is a cytokine that has been implicated in the pathogenesis of several malignancies, including PCa. Two IL-6 gene polymorphisms, - 174 G/C (rs1800795) and - 572 C/G (rs1800796), have received intellectual attention due to their potential role as modulators of prostate cancer risk. The main objective of this research was to comprehensively explore the potential associations between IL-6 rs1800795 and rs1800796 polymorphisms, and their impact on the occurrence of PCa. A case-control study was carried out with a well-defined cohort comprising 110 PCa cases and 110 controls (total n = 220). The genotyping of rs1800795 and rs1800796 was carefully performed using the highly sensitive and accurate Polymerase Chain Reaction-High Resolution Melting Curve (PCR-HRM) technique. The assessment of genetic associations was evaluated using various R packages, such as Haplo-Stats, SNP stat, pheatmap, and LD heatmap. The present study applied odds ratio (OR) analysis to reveal significant evidence of strong associations between the genotypes of rs1800795 and rs1800796 and the susceptibility to PCa. The findings of this study underscore the noteworthy impact of genetic variations in the IL-6 gene on the development of prostate cancer. Specifically, the C/G and G/G genotypes of rs1800795 demonstrated increased PCa risk, with odds ratios (OR) of 1.650 (95% CI = 1.068-2.549, p = 0.032) and 2.475 (95% CI = 1.215-5.043, p < 0.001), respectively. Similarly, the G/C genotype of rs1800796 exhibited an OR of 2.374 (95% CI = 1.363-4.130, p = 0.012) for elevated prostate cancer risk, while the C/C genotype had an OR of 1.81 (95% CI = 1.02-3.22, p = 0.7). Furthermore, our haplotype analysis have revealed an association between haplotype 4 (C-G) and increased risk of PCa (OR = 1.69, 95% CI = 1.05-2.73, p = 0.032). In conclusion, this case-control analysis presents compelling evidence for a significant association between IL-6 variants (rs1800795 and rs1800796) and increased susceptibility to prostate cancer.

Predictive value of single-nucleotide polymorphism signature for nephrolithiasis recurrence: a 5-year prospective study.

Background: Genetic variations are linked to kidney stone formation. However, the association of single nucleotide polymorphism (SNPs) and stone recurrence has not been well studied. This study aims to identify genetic variants associated with kidney stone recurrences and to construct a predictive nomogram model using SNPs and clinical features to predict the recurrence risk of kidney stones. Methods: We genotyped 49 SNPs in 1001 patients who received surgical stone removal between Jan 1 and Dec 31 of 2012. All patients were confirmed stone-free by CT scan and then received follow-up at least 5 years. SNP associations with stone recurrence were analyzed by Cox proportion hazard model. A predictive nomogram model using SNPs and clinical features to predict the recurrence risk of kidney stones was developed by use of LASSO Cox regression. Results: The recurrence rate at 3, 5, 7 years were 46.8%, 71.2%, and 78.4%, respectively. 5 SNPs were identified that had association with kidney stone recurrence risk. We used computer-generated random numbers to assign 500 of these patients to the training cohort and 501 patients to the validation cohort. A nomogram that combined the 14-SNPs-based classifier with the clinical risk factors was constructed. The areas under the curve (AUCs) at 3, 5 and 7 years of this nomogram was 0.645, 0.723, and 0.75 in training cohort, and was 0.631, 0.708, and 0.727 in validation cohort, respectively. Results show that the nomogram presented a higher predictive accuracy than those of the SNP classifier or clinical factors alone. Conclusion: SNPs are significantly associated with kidney stone recurrence and should add prognostic value to the traditional clinical risk factors used to assess the kidney stone recurrence. A nomogram using clinical and genetic variables to predict kidney stone recurrence has revealed its potential in the future as an assessment tool during the follow-up of kidney stone patients.

A critical appraisal of urolithiasis clinical practice guidelines using the AGREE II instrument.

Background: The Appraisal of Guidelines for Research and Evaluation II (AGREE II) instrument was developed to improve the methodological quality of clinical practice guidelines (CPGs). High-quality guidelines can provide reliable recommendations for different clinical issues. Currently, there is no quality appraisal of CPGs for urolithiasis. This study evaluated the quality of evidence-based CPGs for urolithiasis and provided new insights into improving guideline quality on urolithiasis. Methods: Systematic reviews were conducted to identify urolithiasis CPGs in PubMed, electronic databases, and websites of medical associations from January 2009 to July 2022. The quality of included CPGs was evaluated by four reviewers using the AGREE II instrument. Subsequently, the scores of all domains in the AGREE II instrument were calculated. Results: A total of 19 urolithiasis CPGs were identified for review: seven from Europe, six from USA, three from international union, two from Canada, and one from Asia. The agreement among reviewers was rated good [intraclass correlation coefficient (ICC), 0.806; 95% CI: 0.779-0.831]. The domains with the highest scores were scope and purpose (69.7%, 54.2-86.1%) and clarity of presentation (76.8%, 59.7-90.3%). The domains of stakeholder involvement (44.9%, 19.4-84.7%) and applicability (48.5%, 30.2-72.9%) gained the lowest score. Only five guidelines (26.3%) were considered "strongly recommended". Conclusions: The overall quality of the eligible CPGs was relatively high; however, future work is still needed in the domains of rigor of development, editorial independence, applicability, and stakeholder involvement.

Extracellular vesicle-circEHD2 promotes the progression of renal cell carcinoma by activating cancer-associated fibroblasts.

BACKGROUND: The encapsulation of circular RNAs (circRNAs) into extracellular vesicles (EVs) enables their involvement in intercellular communication and exerts an influence on the malignant advancement of various tumors. However, the regulatory role of EVs-circRNA in renal cell carcinoma (RCC) remains elusive. METHODS: The in vitro and in vivo functional experiments were implemented to measure the effects of circEHD2 on the phenotype of RCC. The functional role of EVs-circEHD2 on the activation of fibroblasts was assessed by collagen contraction assay, western blotting, and enzyme-linked immunosorbent assay (ELISA). The mechanism was investigated by RNA pull-down assay, RNA immunoprecipitation, chromatin isolation by RNA purification, luciferase assay, and co-immunoprecipitation assay. RESULTS: We demonstrated that circEHD2 was upregulated in RCC tissues and serum EVs of RCC patients with metastasis. Silencing circEHD2 inhibited tumor growth in vitro and in vivo. Mechanistic studies indicated that FUS RNA -binding protein (FUS) accelerated the cyclization of circEHD2, then circEHD2 interacts with tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein eta (YWHAH), which acts as a bridge to recruit circEHD2 and Yes1-associated transcriptional regulator (YAP) to the promoter of SRY-box transcription factor 9 (SOX9); this results in the sustained activation of SOX9. Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1) regulates the package of circEHD2 into EVs, then EVs-circEHD2 transmits to fibroblasts, converting fibroblasts to cancer-associated fibroblasts (CAFs). Activated CAFs promote the metastasis of RCC by secreting pro-inflammatory cytokines such as IL-6. Furthermore, antisense oligonucleotides (ASOs) targeting circEHD2 exhibited a strong inhibition of tumor growth in vivo. CONCLUSIONS: The circEHD2/YWHAH/YAP/SOX9 signaling pathway accelerates the growth of RCC. EVs-circEHD2 facilitates the metastasis of RCC by converting fibroblasts to CAFs. Our results suggest that EVs-circEHD2 may be a useful biomarker and therapeutic target for RCC.

[Corrigendum] miR­195 inhibits cell proliferation and angiogenesis in human prostate cancer by downregulating PRR11 expression.

Subsequently to the publication of the above paper, an interested reader drew to the authors' attention that a pair of data panels featured in Figs. 1B and 4C contained overlapping sections, such that data that were intended to show the results from differently performed experiments appeared to have been derived from the same original source (specifically, the 'LNCaP / miR­NC' panel in Fig. 1B and the 'LNCaP / miR­195+ PRR11' panel in Fig. 4C were overlapping). The authors were able to re­examine their original data files, and realized that this figure had been inadverently assembled incorrectly. The revised version of Fig. 1, containing the correct data for Fig. 1B (wherein the error was contained), is shown on the next page. Note that the revisions made to this figure do not affect the overall conclusions reported in the paper. The authors are grateful to the Editor of Oncology Reports for allowing them the opportunity to publish this Corrigendum, and apologize to the readership for any inconvenience caused. [Oncology Reports 39: 1658­1670, 2018; DOI: 10.3892/or.2018.6240].

Designing Intelligent Nanomaterials to Achieve Highly Sensitive Diagnoses and Multimodality Therapy of Bladder Cancer.

Bladder cancer (BC) is among the most common malignant tumors of the genitourinary system worldwide. In recent years, the rate of BC incidence has increased, and the recurrence rate is high, resulting in poor quality of life for patients. Therefore, how to develop an effective method to achieve synchronous precise diagnoses and BC therapies is a difficult problem to solve clinically. Previous reports usually focus on the role of nanomaterials as drug delivery carriers, while a summary of the functional design and application of nanomaterials is lacking. Summarizing the application of functional nanomaterials in high-sensitivity diagnosis and multimodality therapy of BC is urgently needed. This review summarizes the application of nanotechnology in BC diagnosis, including the application of nanotechnology in the sensoring of BC biomarkers and their role in monitoring BC. In addition, conventional and combination therapies strategy in potential BC therapy are analyzed. Moreover, different kinds of nanomaterials in BC multimodal therapy according to pathological features of BC are also outlined. The goal of this review is to present an overview of the application of nanomaterials in the theranostics of BC to provide guidance for the application of functional nanomaterials to precisely diagnose and treat BC.

Accurate diagnosis of prostate cancer with CRISPR-based nucleic acid test strip by simultaneously identifying PCA3 and KLK3 genes.

Although serum prostate specific antigen (PSA) testing could decrease the morality of prostate cancer (PCa), its low specificity usually led to misdiagnosis due to prostatitis or benign prostatic hyperplasia (BPH). Prostate cancer antigen 3 (PCA3) as an alternative prostate tumor-specificity biomarker could be used to increase the specificity of PCa diagnosis, however, it usually required sophisticated operation and expensive equipment for routine detection. Herein, we constructed an early detection platform for prostate cancer with reverse transcriptase-recombinase aided amplification (RT-RAA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 based nucleic acid test strip. The amplicons of PCA3 and kallikrein related peptidase 3 (KLK3) gene, which amplified simultaneously by single-amplification unit of RT-RAA were specifically recognized by Cas9-sgRNA and visual on the nucleic acid test strip by naked eyes without instruments. Simultaneously detection of PCA3 and KLK3 gene could improve specificity and accuracy of the diagnosis but avoid mutual interference. In addition, the platform presented a detection limit of 500 fg/µL and 50 fg/µL in PCA3 and KLK3 gene, respectively. Furthermore, the analysis result of signal ratio of PCA3 to KLK3 gene of urine and peripheral blood specimens from 32 men with suspected prostate cancer on test strips illustrated that the area under the curve values of urine and peripheral blood specimens were 0.998 and 1.0 respectively. In summary, our study highlighted a facile strategy to design an accurate prostate cancer gene detection platform which had the potential to conduct prostate cancer early detection in the resource-limited or other point-of-care testing (POCT) environments.

Fc Fragment of IgE Receptor Ig (FCER1G) acts as a key gene involved in cancer immune infiltration and tumour microenvironment.

Although recent studies have revealed the relationship between Fc Fragment of IgE Receptor Ig (FCER1G) and human tumours, there is still a lack of a more comprehensive pan-cancer analysis of FCER1G as an immune-related gene. In this study, we investigated the expression pattern and prognostic value of FCER1G based on multiple databases. Subsequently, we further explored the role of FCER1G in tumour proliferation and metastasis, as well as its genomic alterations and DNA methylation levels, we next assessed the association between FCER1G and the immune infiltrating cells of the tumour microenvironment in different cancers and verified it by immunohistochemical staining. The correlation between FCER1G and immune checkpoint genes expression and its predictive power in the immune checkpoint blockade treatment cohorts were used to evaluate the importance of FCER1G in immunotherapy. Enrichment analysis of FCER1G-associated partners was also performed. In addition, we substantiated the expression of FCER1G in specific cell types of different tumours using single-cell RNA sequencing data from different databases. Our research results showed that FCER1G is up-regulated in most tumour. Positive associations were found between FCER1G expression and tumour prognosis, proliferation, and metastasis, we also found that FCER1G is closely related to the tumour microenvironment and tumour immunity. Moreover, FCER1G-associated partners were enriched in pathways associated with neutrophils activation. Finally, we confirmed that FCER1G was mainly expressed in monocyte/macrophages of the tumour microenvironment. In conclusion, our findings provided a comprehensive understanding of FCER1G in oncogenesis and tumour immunology among various tumours and demonstrated its potential value in prognosis prediction and tumour immunotherapy.

Cerium oxide-based nanozyme suppresses kidney calcium oxalate crystal depositions via reversing hyperoxaluria-induced oxidative stress damage.

Oxidative stress damage to renal epithelial cells is the main pathological factor of calcium oxalate calculi formation. The development of medicine that could alleviate oxidative damage has become the key to the prevention and treatment of urolithiasis. Herein, porous nanorods CeO2 nanoparticles (CNPs) were selected from CeO2 with different morphologies as an antioxidant reagent to suppress kidney calcium oxalate crystal depositions with excellent oxidation resistance due to its larger specific surface area. The reversible transformation from Ce3+ to Ce4+ could catalyze the decomposition of excess free radicals and act as a biological antioxidant enzyme basing on its strong ability to scavenge free radicals. The protection capability of CNPS against oxalate-induced damage and the effect of CNPS on calcium oxalate crystallization were studied. CNPS could effectively reduce reactive oxygen species production, restore mitochondrial membrane potential polarity, recover cell cycle progression, reduce cell death, and inhibit the formation of calcium oxalate crystals on the cell surface in vitro. The results of high-throughput sequencing of mRNA showed that CNPs could protect renal epithelial cells from oxidative stress damage caused by high oxalate by suppressing the expression gene of cell surface adhesion proteins. In addition, CNPS can significantly reduce the pathological damage of renal tubules and inhibit the deposition of calcium oxalate crystals in rat kidneys while having no significant side effect on other organs and physiological indicators in vivo. Our results provide a new strategy for CNPS as a potential for clinical prevention of crystalline kidney injury and crystal deposition.

Distinct immune and inflammatory response patterns contribute to the identification of poor prognosis and advanced clinical characters in bladder cancer patients.

Due to the molecular heterogeneity, most bladder cancer (BLCA) patients show no pathological responses to immunotherapy and chemotherapy yet suffer from their toxicity. This study identified and validated three distinct and stable molecular clusters of BLCA in cross-platform databases based on personalized immune and inflammatory characteristics. H&E-stained histopathology images confirmed the distinct infiltration of immune and inflammatory cells among clusters. Cluster-A was characterized by a favorable prognosis and low immune and inflammatory infiltration but showed the highest abundance of prognosis-related favorable immune cell and inflammatory activity. Cluster-B featured the worst prognosis and high immune infiltration, but numerous unfavorable immune cells exist. Cluster-C had a favorable prognosis and the highest immune and inflammatory infiltration. Based on machine learning, a highly precise predictive model (immune and inflammatory responses signature, IIRS), including FN1, IL10, MYC, CD247, and TLR2, was developed and validated to identify the high IIRS-score group that had a poor prognosis and advanced clinical characteristics. Compared to other published models, IIRS showed the highest AUC in 5 years of overall survival (OS) and a favorable predictive value in predicting 1- and 3- year OS. Moreover, IIRS showed an excellent performance in predicting immunotherapy and chemotherapy's response. According to immunohistochemistry and qRT-PCR, IIRS genes were differentially expressed between tumor tissues with corresponding normal or adjacent tissues. Finally, immunohistochemical and H&E-stained analyses were performed on the bladder tissues of 13 BLCA patients to further demonstrate that the IIRS score is a valid substitute for IIR patterns and can contribute to identifying patients with poor clinical and histopathology characteristics. In conclusion, we established a novel IIRS depicting an IIR pattern that could independently predict OS and acts as a highly precise predictive biomarker for advanced clinical characters and the responses to immunotherapy and chemotherapy.

ATR Contributes More Than ATM in Intra-S-Phase Checkpoint Activation after IR, and DNA-PKcs Facilitates Recovery: Evidence for Modular Integration of ATM/ATR/DNA-PKcs Functions.

The intra-S-phase checkpoint was among the first reported cell cycle checkpoints in mammalian cells. It transiently slows down the rate of DNA replication after DNA damage to facilitate repair and thus prevents genomic instability. The ionizing radiation (IR)-induced intra-S-phase checkpoint in mammalian cells is thought to be mainly dependent upon the kinase activity of ATM. Defects in the intra-S-phase checkpoint result in radio-resistant DNA synthesis (RDS), which promotes genomic instability. ATM belongs to the PI3K kinase family along with ATR and DNA-PKcs. ATR has been shown to be the key kinase for intra-S-phase checkpoint signaling in yeast and has also been implicated in this checkpoint in higher eukaryotes. Recently, contributions of DNA-PKcs to IR-induced G2-checkpoint could also be established. Whether and how ATR and DNA-PKcs are involved in the IR-induced intra-S-phase checkpoint in mammalian cells is incompletely characterized. Here, we investigated the contributions of ATM, ATR, and DNA-PKcs to intra-S-phase checkpoint activation after exposure to IR of human and hamster cells. The results suggest that the activities of both ATM and ATR are essential for efficient intra-S-phase checkpoint activation. Indeed, in a wild-type genetic background, ATR inhibition generates stronger checkpoint defects than ATM inhibition. Similar to G2 checkpoint, DNA-PKcs contributes to the recovery from the intra-S-phase checkpoint. DNA-PKcs-deficient cells show persistent, mainly ATR-dependent intra-S-phase checkpoints. A correlation between the degree of DSB end resection and the strength of the intra-S-phase checkpoint is observed, which again compares well to the G2 checkpoint response. We conclude that the organization of the intra-S-phase checkpoint has a similar mechanistic organization to that of the G2 checkpoint in cells irradiated in the G2 phase.