DNA Damage Checkpoint Control Research Articles

Mechanistic insights into DNA damage recognition and checkpoint control in plants.

The plant DNA damage response (DDR) pathway safeguards genomic integrity by rapid recognition and repair of DNA lesions that, if unrepaired, may cause genome instability. Most frequently, DNA repair goes hand in hand with a transient cell cycle arrest, which allows cells to repair the DNA lesions before engaging in a mitotic event, but consequently also affects plant growth and yield. Through the identification of DDR proteins and cell cycle regulators that react to DNA double-strand breaks or replication defects, it has become clear that these proteins and regulators form highly interconnected networks. These networks operate at both the transcriptional and post-transcriptional levels and include liquid-liquid phase separation and epigenetic mechanisms. Strikingly, whereas the upstream DDR sensors and signalling components are well conserved across eukaryotes, some of the more downstream effectors are diverged in plants, probably to suit unique lifestyle features. Additionally, DDR components display functional diversity across ancient plant species, dicots and monocots. The observed resistance of DDR mutants towards aluminium toxicity, phosphate limitation and seed ageing indicates that gaining knowledge about the plant DDR may offer solutions to combat the effects of climate change and the associated risk for food security.

Abstract 2276: High frequency of pathogenic germline variants in patients with malignant mesothelioma

Abstract Background: Malignant mesothelioma (MM) is associated with asbestos exposure, tumor heterogeneity and aggressive clinical behavior. Identification and characterization of germline variants in malignant mesothelioma is relevant for identifying potential actionable targets and genetic counseling of family members. Methods: Patients referred to the Phase I Unit at Rigshospitalet were prospectively enrolled into the Copenhagen Prospective Personalized Oncology trial and underwent whole exome sequencing for somatic and germline variants. Between January 2014 and September 2021, 45 patients with MM were identified and 40 patients underwent whole exome sequencing. Germline variants were selected according to association to inherited cancer using a 168-gene panel and variants were classified according to ACMG/AMP classification as pathological (class 5) or likely pathogenic (class 4). Results: 34 males (85%) and 6 females (15%) with a median age of 64 years (range: 43-77) were available for analysis. The majority (85%) had malignant pleural mesothelioma whilst the rest (15%) had malignant peritoneal mesothelioma. Histological subtypes were distributed between epithelioid type (N=24, 60%), biphasic type (N=12, 30%) and the sarcomatoid type (N=4, 10%). A pathogenic or likely pathogenic variant was found in 16 patients (40%). In total 13 different germline variants were identified (ATM, BAP1, BRCA2, CDKN2A, FANCA, FANCC, FANCD2, FANCM, MUTYH, NBN, RAD51B, SDHA and XPC). The variants included five frameshifts (33%), four missenses (25%), four nonsenses (25%), one splice site (6%), one start loss (6%) and one synonymous (6%). The pathogenic germline variants were found in DNA repair pathways, including homologous recombination repair (69%), nucleotide excision repair (13%), cell cycle regulatory (6%), DNA damage checkpoint control (6%) and hypoxic pathway (6%). Three (19%) of the patients with a germline variant had a second cancer. A previously undiagnosed BRCA2 germline mutation was found in 2 patients. A potential treatment target based on the pathogenic germline variant could be suggested in four patients (10%). Conclusion: The current analysis revealed a high frequency of pathogenic germline variants in patients with malignant mesothelioma. These data support germline testing in these patients and provide a rationale for additional investigation of the homologous recombination pathway as a potential target for precision medicine. Citation Format: Laila Belcaid, Birgitte Bertelsen, Karin A. Wadt, Ida V. Tuxen, Iben Spanggaard, Martin Højgaard, Jens B. Sørensen, Ulrik Lassen, Finn C. Nielsen, Kristoffer Rohrberg, Christina W. Yde. High frequency of pathogenic germline variants in patients with malignant mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2276.

MDC1 is essential for G2/M transition and spindle assembly in mouse oocytes.

Mammalian oocytes are particularly susceptible to accumulating DNA damage. However, unlike mitotic cells in which DNA damage induces G2 arrest by activating the ATM-Chk1/2-Cdc25 pathway, oocytes readily enter M-phase immediately following DNA damage. This implies a lack of a robust canonical G2/M DNA damage checkpoint in oocytes. Here we show that MDC1 plays a non-canonical role in controlling G2/M transition by regulating APC/C-Cdh1-mediated cyclin B1 degradation in response to DNA damage in mouse oocytes. Depletion of MDC1 impaired M-phase entry by decreasing cyclin B1 levels via the APC/C-Cdh1 pathway. Notably, the APC/C-Cdh1 regulation mediated by MDC1 was achieved by a direct interaction between MDC1 and APC/C-Cdh1. This interaction was transiently disrupted after DNA damage with a concomitant increase in Cdh1 levels, which, in turn, decreased cyclin B1 levels and delayed M-phase entry. Moreover, MDC1 depletion impaired spindle assembly by decreasing the integrity of microtubule organizing centers (MTOCs). Therefore, our results demonstrate that MDC1 is an essential molecule in regulating G2/M transition in response to DNA damage and in regulating spindle assembly in mouse oocytes. These results provide new insights into the regulation of the G2/M DNA damage checkpoint and cell cycle control in oocytes.

Checkpoint kinases are required for oocyte meiotic progression by the maintenance of normal spindle structure and chromosome condensation

Checkpoint kinases (Chk) 1/2 are known for DNA damage checkpoint and cell cycle control in somatic cells. According to recent findings, the involvement of Chk1 in oocyte meiotic resumption and Chk2 is regarded as an essential regulator for progression at the post metaphase I stage (MI). In this study, AZD7762 (Chk1/2 inhibitor) and SB218078 (Chk1 inhibitor) were used to uncover the joint roles of Chk1/2 and differentiate the importance of Chk1 and Chk2 during oocyte meiotic maturation. Inhibition of Chk1/2 or Chk1 alone had no significant effect on germinal vesicle breakdown (GVBD) but significantly inhibited the first polar body (PB1). Interestingly, inhibition of Chk1 alone could not increase or completely block the extrusion of PB1 like Chk1/2 inhibition. Also, Chk1/2 inhibition resulted in defective meiotic spindle organization and chromosome condensation both in MI and metaphase II (MII) stages of oocytes. The location of γ-tubulin and Securin were abnormal or missing, while P38 MAPK was activated by Chk1/2 inhibition. Meanwhile, Chk1/2 inhibition reduced the percentage of the second polar body extrusion and pronuclear formation. In conclusion, our results further understand the functions and regulatory mechanism of Chk1/2 during oocyte meiotic maturation.

Tumor suppressive role of mitochondrial sirtuin 4 in induction of G2/M cell cycle arrest and apoptosis in hepatitis B virus-related hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is developed from uncontrolled cell growth after the malignant transformation of hepatocytes. The hepatitis B virus (HBV) X protein (HBx) has shown to induce cell cycle progression and hepatocarcinogenesis. A sub-fraction of HBx is localized in the mitochondria. Sirtuin 4 (SIRT4), a mitochondrial protein, has been demonstrated to play a tumor-suppressive role in many cancers, including HCC. However, little is known about the association between mitochondrial HBx and SIRT4 during hepatocarcinogenesis. We aimed to investigate the clinical significance and functional role of SIRT4 in HBV-related HCC. SIRT4 expression was significantly lower in the HCC tissues collected from 30 patients with HBV-related HCC than in normal liver tissues from control patients (p < 0.0001). TCGA data analysis indicated that SIRT4 expression was also lower in patients with HBV infection than in those without, and SIRT4 levels were positively associated with better patient survival. Similarly, HCC cell lines had lower SIRT4 expression than normal liver cell lines (all p < 0.01). Among the HCC cell lines, those harbored HBV had a lower SIRT4 expression than those without HBV (p < 0.0001). In vitro experiments revealed that stable HBx transfection suppressed SIRT4 expression in both HepG2 and Huh7 cells (both p < 0.001). Ectopic SIRT4 overexpression alone could induce cellular senescence through arresting cell-cycle progression at G2/M, and inducing cell apoptosis in HCC cells. Mechanistically, SIRT4 upregulated cell-cycle governing genes p16 and p21 protein expression, suppressed CyclinB1/Cdc2 and Cdc25c which normally induce cell-cycle progression, and suppressed survivin to induce apoptosis. Our findings demonstrate the interaction between HBV and SIRT4 in the context of HCC. SIRT4 involves in G2/M DNA damage checkpoint control and genomic stability in hepatocarcinogenesis, which could be targeted for future anticancer strategies.

Robust and Tunable Toggle Switches with Interlocked Positive Feedback Loops

Toggle switches are widely used to make critical all-or-none decisions such as cell differentiation or apoptosis. While the toggle switch can be generated by using a single-type positive feedback loop (PFL), a natural toggle switch frequently emerges from interlocked PFLs composed of two different types of PFLs: one with two mutually activated units and the other with two mutually inhibiting units. To investigate the advantage of the interlocked PFL over the single type of PFL, here, we carefully analyze how the interlocked PFLs generate bistability and thus the toggle switch. We find that in the interlocked PFLs, mutual activation and mutual inhibition cooperate to generate a robust merged bistability. Interestingly, we also find that turning-on and turning-off of the merged bistability can be flexibly controlled by using the balance between the strengths of two competing PFLs. We illustrate the role of such robust and tunable joint toggle switches of the interlocked PFLs in the DNA damage checkpoint control of G2/M transition. Our work describes how natural toggle switches achieve two critical properties, robustness and tunability, simultaneously by merging opposite types of PFLs.

ER\u03b1-related chromothripsis enhances concordant gene transcription on chromosome 17q11.1-q24.1 in luminal breast cancer

BackgroundChromothripsis is an event of genomic instability leading to complex chromosomal alterations in cancer. Frequent long-range chromatin interactions between transcription factors (TFs) and targets may promote extensive translocations and copy-number alterations in proximal contact regions through inappropriate DNA stitching. Although studies have proposed models to explain the initiation of chromothripsis, few discussed how TFs influence this process for tumor progression.MethodsThis study focused on genomic alterations in amplification associated regions within chromosome 17. Inter−/intra-chromosomal rearrangements were analyzed using whole genome sequencing data of breast tumors in the Cancer Genome Atlas (TCGA) cohort. Common ERα binding sites were defined based on MCF-7, T47D, and MDA-MB-134 breast cancer cell lines using univariate K-means clustering methods. Nanopore sequencing technology was applied to validate frequent rearrangements detected between ATC loci on 17q23 and an ERα hub on 20q13. The efficacy of pharmacological inhibition of a potentially druggable target gene on 17q23 was evaluated using breast cancer cell lines and patient-derived circulating breast tumor cells.ResultsThere are five adjoining regions from 17q11.1 to 17q24.1 being hotspots of chromothripsis. Inter−/intra-chromosomal rearrangements of these regions occurred more frequently in ERα-positive tumors than in ERα-negative tumors. In addition, the locations of the rearrangements were often mapped within or close to dense ERα binding sites localized on these five 17q regions or other chromosomes. This chromothriptic event was linked to concordant upregulation of 96 loci that predominantly regulate cell-cycle machineries in advanced luminal tumors. Genome-editing analysis confirmed that an ERα hub localized on 20q13 coordinately regulates a subset of these loci localized on 17q23 through long-range chromosome interactions. One of these loci, Tousled Like Kinase 2 (TLK2) known to participate in DNA damage checkpoint control, is an actionable target using phenothiazine antipsychotics (PTZs). The antiproliferative effect of PTZs was prominent in high TLK2-expressing cells, compared to low expressing cells.ConclusionThis study demonstrates a new approach for identifying tumorigenic drivers from genomic regions highly susceptible to ERα-related chromothripsis. We found a group of luminal breast tumors displaying 17q-related chromothripsis for which antipsychotics can be repurposed as treatment adjuncts.

Abstract P4-05-17: ESR1 related chromothripsis enhances concordant gene transcription on chromosome 17q11.1-q24.1 in luminal breast cancer

Abstract Chromothripsis is an event of genomic instability leading to complex chromosomal alterations in cancer. Although studies have proposed models to explain its initiation, few discussed how transcription factors (TFs) influence chromothripsis for tumor progression. Frequent long-range chromatin interactions between TFs and targets may promote extensive translocations and copy-number alterations in proximal contact regions through inappropriate DNA stitching. Herein we surveyed genomic alterations of chromosome 17 in 1,014 breast tumors in the Cancer Genome Atlas (TCGA) cohort and found five adjoining regions from 17q11.1 to 17q24.1 being hotspots of chromothripsis. Inter-/intra-chromosomal rearrangements of these regions occurred more frequently in ERα-positive tumors than in ERα-negative tumors. In addition, the rearrangement sites were often mapped within or close to dense estrogen receptor 1 (ESR1) sites localized on the regions or other chromosomes. This chromothriptic event was linked to concordant upregulation of 96 loci that predominantly regulate cell-cycle machineries in advanced luminal tumors. Genome-editing analysis confirmed that an ESR1 transcription hub localized on 20q13 coordinately regulates a subset of these loci localized on 17q23 through long-range chromosome interactions. One of these loci, Tousled Like Kinase 2 (TLK2) known to participate in DNA damage checkpoint control, is an actionable target using phenothiazine antipsychotics (PTZs). The antiproliferative effect of PTZs was prominent in high TLK2-expressing cells, compared to low expressing cells. Collectively, we identified a group of luminal breast tumors displaying 17q-related chromothripsis for which antipsychotics can be repurposed as treatment adjuncts. Citation Format: Chun-Lin Lin. ESR1 related chromothripsis enhances concordant gene transcription on chromosome 17q11.1-q24.1 in luminal breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-05-17.

PALB2 connects BRCA1 and BRCA2 in the G2/M checkpoint response.

The G2/M checkpoint inhibits mitotic entry upon DNA damage thereby preventing segregation of broken chromosomes and preserving genome stability. The tumor suppressor proteins BRCA1, PALB2 and BRCA2 constitute a BRCA1-PALB2-BRCA2 axis that is essential for homologous recombination (HR)-based DNA double strand break repair. Besides HR, BRCA1 has been implicated in both the initial activation and the maintenance of the G2/M checkpoint, while BRCA2 and PALB2 have been shown to be critical for its maintenance. Here we show that all 3 proteins can play a significant role in both checkpoint activation and checkpoint maintenance, depending on cell type and context, and that PALB2 links BRCA1 and BRCA2 in checkpoint response. The BRCA1-PALB2 interaction can be important for checkpoint activation, whereas the PALB2-BRCA2 complex formation appears to be more critical for checkpoint maintenance. Interestingly, the function of PALB2 in checkpoint response appears to be independent of CHK1 and CHK2 phosphorylation. Following ionizing radiation, cells with disengaged BRCA1-PALB2 interaction show greatly increased chromosomal abnormalities due apparently to combined defects in HR and checkpoint control. These findings provide new insights into DNA damage checkpoint control and further underscore the critical importance of the proper cooperation of the BRCA and PALB2 proteins in genome maintenance.

OsRAD17 Is Required for Meiotic Double-Strand Break Repair and Plays a Redundant Role With OsZIP4 in Synaptonemal Complex Assembly.

The repair of SPO11-dependent double-strand breaks (DSBs) by homologous recombination (HR) ensures the correct segregation of homologous chromosomes. In yeast and human, RAD17 is involved in DNA damage checkpoint control and DSB repair. However, little is known about its function in plants. In this study, we characterized the RAD17 homolog in rice. In Osrad17 pollen mother cells (PMCs), associations between non-homologous chromosomes and chromosome fragmentation were constantly observed. These aberrant chromosome associations were dependent on the formation of programmed DSBs. OsRAD17 interacts with OsRAD1 and the meiotic phenotype of Osrad1 Osrad17 is indistinguishable from the two single mutants which have similar phenotypes, manifesting they could act in the same pathway. OsZIP4, OsMSH5 and OsMER3 are members of ZMM proteins in rice that are required for crossover formation. We found that homologous pairing and synapsis, which was roughly unaffected in Oszip4 and Osrad17 single mutant, was severely disturbed in the Oszip4 Osrad17 double mutant. Similar phenotypes were observed in the Osmsh5 Osrad17 and Osmer3 Osrad1 double mutants, suggesting the cooperation between the checkpoint proteins and ZMM proteins in assuring accurate HR in rice.

PO-133 Inhibition of HIF-1α enhances radiation sensitivity in tumour cells and mediates tumour growth decrease in mice

IntroductionPoor prognosis of many solid tumours is often associated with hypoxic regions and an increased level of hypoxia-inducible-factor-1α (HIF-1α). Previous findings indicate that HIF-1α expression is relevant for radiation resistance. It was shown that HIF-1α is connected to molecules involved in DNA damage repair and checkpoint control. HIF-1α has an enhanced activity in tumours after radiation treatment (IR) and also mediates low radiation sensitivity.Material and methodsIn vitro cultured Lewis Lung Carcinoma (LLC) cells with doxycycline (Dox)-inducible knockdown (KD) of HIF-1α were used. Cells were incubated under hypoxic conditions (Hx, 1%) and irradiated with 5 Gy. Cell cycle distribution analysis was performed by PI staining and FACS. Colony formation assay, caspase-3 assay, PARP-1 cleavage and Annexin/PI staining were used to quantify apoptosis and long term survival. DNA double strand break (DSB) repair was investigated by pulse field gel electrophoresis, Western blot and immunofluorescent staining with γH2AX, 53 BP1, RAD51, BRCA1 antibodies. In vivo the induction of HIF-1α KD was performed by tamoxifen administration to HIF-1αfl/flCre-ERki/+ C57BL/6 mice. The LLC cells with constitutive HIF-1α targeting (shHIF) or scrambled (shscr) shRNA-expressing vectors were inoculated into the flank of the mice. IR was delivered once at day 10 with a dose of 15 Gy using an X-ray source.Results and discussionsWe demonstrated an inhibition of decrement of phosphorylated H2AX after Hx and IR in HIF-1α deficient cells. Fast repair kinetics of the cells remained unchanged, whereas the long term survival of the cells with reduced HIF-1α was decreased. Moreover, these cells displayed an increased rate of apoptosis after IR. We also observed persisted RAD51 foci after 24 hour in HIF-1α deficient cells which indicates an alteration in homologous recombination repair (HRR) of the cells. Furthermore, in vivo experiments with a HIF-1α deficient stromal cells in the mice and injected HIF-1α deficient tumours cells indicate a decrease of tumour growth.ConclusionThese results imply that inactivation of HIF-1α disrupts DSB repair, in particular HRR. Furthermore, they highlight the importance of the interaction of tumour and microenvironment with respect to radiation sensitivity. It was shown that inhibition of HIF-1α enhances radiation sensitivity of tumour cells which is potentially helpful for the development of novel tumour therapies.

Abstract 2026: CHK1 inhibition synergizes with cisplatin to promote mitotic cell death via activation of caspase 2 and downregulation of E2F1 in small cell lung cancer

Abstract Platinum-based chemotherapy remains the standard treatment for patients with small cell lung cancer (SCLC), one of the deadliest cancers, but the benefit of such therapy is often hampered by rapid emergence of drug resistance. Thus far, there is no targeted therapy available for SCLC, mainly due to the lack of targetable oncogenic driver alterations; nevertheless, over 90% of SCLC tumors harbor inactivating mutations in tumor suppressor p53, an important checkpoint regulator for the control of cell cycle progression in the event of DNA damage. SCLC cells with p53-deficiency heavily rely on the checkpoint kinase 1 (CHK1), a key checkpoint regulator, to prevent the progression of cell cycle through G2/M in the presence of damaged DNA. Using CHK1 inhibitors and siRNA knockdown approaches, we explored the potential therapeutic benefit of CHK1 inhibition in SCLC models in combination with cisplatin, a DNA damaging agent. We demonstrated that CHK1 inhibition was able to release G2/M cell cycle arrest induced by cisplatin, and synergized with cisplatin to induce mitotic cell death especially in the p53-deficient SCLC cell lines. CHK1 inhibition activated caspase 2 and downregulated the expression of E2F1 transcription factor in these cells. We showed that activation of caspase 2 was required for the mitotic cell death induced by the combination of cisplatin and CHK1 inhibition. Interestingly, the upregulation of E2F1 induced by CHK1 inhibition was mediated in part by caspase 2, and E2F1 played a role in the DNA damage checkpoint control as knockdown of E2F1 enhanced cytotoxicity induced by cisplatin or CHK1 inhibitor in GLC4 cells. However, ectopic overexpression of E2F1 also led to increase of phospho-histone H3, γH2AX and cleaved PARP, and significantly enhanced cisplatin cytotoxicity, indicating that proper dosage of E2F1 is critical for DNA damage checkpoint response. These data provide a mechanistic insight of CHK1-mediated DNA damage checkpoint control and the anticancer effect of CHK1 inhibition against SCLC. In tumor samples derived from SCLC patients treated with platinum based chemotherapy, we found that high expression of CHK1 and E2F1 significantly correlated with poorer overall survival, further suggesting a link between CHK1 and E2F1 and potentially collaborative roles of these two factors in DNA damage checkpoint response. Moreover, we showed that CHK1 inhibition resensitized cisplatin-resistant cells to cisplatin treatment and induced mitotic cell death via caspase activation in these cells. These data indicate that CHK1 inhibition may enhance the benefit of cisplatin treatment for SCLC and overcome cisplatin resistance. We are presently confirming the in vitro results of CHK1 inhibition in combination with cisplatin treatment in xenograft tumor models in athymic nude mice. Citation Format: Wei-Hsun Hsu, Xiaoliang Zhao, In-Gyu Kim, Guanhua Rao, Justine McCutcheon, Shuo-Tse Hsu, Afshin Dowlati, Yu-Wen Zhang, Giuseppe Giaccone. CHK1 inhibition synergizes with cisplatin to promote mitotic cell death via activation of caspase 2 and downregulation of E2F1 in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2026. doi:10.1158/1538-7445.AM2017-2026

Tip60: Main Functions and Its Inhibitors.

Tip60, the founding member of MYST histone acetyltransferase family, was originally identified as a cellular acetyltransferase protein that interacts with HIV-1 Tat. Tip60 plays roles in many processes such as cellular signaling transmission, DNA damage repair, cell cycle and checkpoint control and apoptosis by acetylating its histone or non-histone substrates. Dysfunction of Tip60 could promote or suppress diseases including different kinds of cancers. Here, main functions and its known inhibitors were summarized.

A dnaN plasmid shuffle strain for rapid in vivo analysis of mutant Escherichia coli β clamps provides insight into the role of clamp in umuDC-mediated cold sensitivity.

The E. coli umuDC gene products participate in two temporally distinct roles: UmuD2C acts in a DNA damage checkpoint control, while UmuD'2C, also known as DNA polymerase V (Pol V), catalyzes replication past DNA lesions via a process termed translesion DNA synthesis. These different roles of the umuDC gene products are managed in part by the dnaN-encoded β sliding clamp protein. Co-overexpression of the β clamp and Pol V severely blocked E. coli growth at 30°C. We previously used a genetic assay that was independent of the ability of β clamp to support E. coli viability to isolate 8 mutant clamp proteins (βQ61K, βS107L, βD150N, βG157S, βV170M, βE202K, βM204K and βP363S) that failed to block growth at 30°C when co-overexpressed with Pol V. It was unknown whether these mutant clamps were capable of supporting E. coli viability and normal umuDC functions in vivo. The goals of this study were to answer these questions. To this end, we developed a novel dnaN plasmid shuffle assay. Using this assay, βD150N and βP363S were unable to support E. coli viability. The remaining 6 mutant clamps, each of which supported viability, were indistinguishable from β+ with respect to umuDC functions in vivo. In light of these findings, we analyzed phenotypes of strains overexpressing either β clamp or Pol V alone. The strain overexpressing β+, but not those expressing mutant β clamps, displayed slowed growth irrespective of the incubation temperature. Moreover, growth of the Pol V-expressing strain was modestly slowed at 30°, but not 42°C. Taken together, these results suggest the mutant clamps were identified due to their inability to slow growth rather than an inability to interact with Pol V. They further suggest that cold sensitivity is due, at least in part, to the combination of their individual effects on growth at 30°C.

Different DNA damage and cell cycle checkpoint control in low‐ and high‐risk human papillomavirus infections of the vulva

Human papillomavirus (HPV) infections may result in benign hyperplasia, caused by low-risk HPV types, or (pre)malignant lesions caused by high-risk HPV types. The molecular basis of this difference in malignant potential is not completely understood. Here, we performed gene profiling of different HPV infected vulvar tissues (condylomata acuminata (n = 5), usual type vulvar intraepithelial neoplasia (uVIN) (n = 9)) and control samples (n = 14) using Affymetrix Human U133A plus 2 GeneChips. Data were analyzed using OmniViz®, Partek® and Ingenuity® Software. Results were validated by real-time RT-PCR and immunostaining. Although similarities were observed between gene expression profiles of low- and high-risk HPV infected tissues (e.g., absence of estrogen receptor in condylomata and uVIN), high-risk HPV infected tissues showed more proliferation and displayed more DNA damage than tissues infected with low-risk HPV. These observations were confirmed by differential regulation of cell cycle checkpoints and by increased expression of DNA damage-biomarkers p53 and γH2AX. Furthermore, FANCA, FANCD2, BRCA1 and RAD51, key players in the DNA damage response, were significantly upregulated (p < 0.05). In addition, we compared our results with publicly available gene expression profiles of various other HPV-induced cancers (vulva, cervix and head-and-neck). This showed p16(INK4a) was the most significant marker to detect a high-risk HPV infection, but no other markers could be found. In conclusion, this study provides insight into the molecular basis of low- and high-risk HPV infections and indicates two main pathways (cell cycle and DNA damage response) that are much stronger affected by high-risk HPV as compared to low-risk HPV.

SET Nuclear Oncogene Associates with Microcephalin/MCPH1 and Regulates Chromosome Condensation

Primary microcephaly is an autosomal recessive disorder characterized by marked reduction in human brain size. Microcephalin (MCPH1), one of the genes mutated in primary microcephaly, plays an important role in DNA damage checkpoint control and mitotic entry. Additionally, MCPH1 ensures the proper temporal activation of chromosome condensation during mitosis, by acting as a negative regulator of the condensin II complex. We previously found that deletion of the of the MCPH1 N terminus leads to the premature chromosome condensation (PCC) phenotype. In the present study, we unexpectedly observed that a truncated form of MCPH1 appears to be expressed in MCPH1(S25X/S25X) patient cells. This likely results from utilization of an alternative translational start codon, which would produce a mutant MCPH1 protein with a small deletion of its N-terminal BRCT domain. Furthermore, missense mutations in the MCPH1 cluster at its N terminus, suggesting that intact function of this BRCT protein-interaction domain is required both for coordinating chromosome condensation and human brain development. Subsequently, we identified the SET nuclear oncogene as a direct binding partner of the MCPH1 N-terminal BRCT domain. Cells with SET knockdown exhibited abnormal condensed chromosomes similar to those observed in MCPH1-deficient mouse embryonic fibroblasts. Condensin II knockdown rescued the abnormal chromosome condensation phenotype in SET-depleted cells. In addition, MCPH1 V50G/I51V missense mutations, impair binding to SET and fail to fully rescue the abnormal chromosome condensation phenotype in Mcph1(-/-) mouse embryonic fibroblasts. Collectively, our findings suggest that SET is an important regulator of chromosome condensation/decondensation and that disruption of the MCPH1-SET interaction might be important for the pathogenesis of primary microcephaly.

The FANC pathway is activated by adenovirus infection and promotes viral replication-dependent recombination

Deciphering the crosstalk between a host cell and a virus during infection is important not only to better define viral biology but also to improve our understanding of cellular processes. We identified the FANC pathway as a helper of viral replication and recombination by searching for cellular targets that are modified by adenovirus (Ad) infection and are involved in its outcome. This pathway, which is involved in the DNA damage response and checkpoint control, is altered in Fanconi anaemia, a rare cancer predisposition syndrome. We show here that Ad5 infection activates the FANC pathway independent of the classical DNA damage response. Infection with a non-replicating Ad shows that the presence of viral DNA is not sufficient to induce the monoubiquitination of FANCD2 but still activates the DNA damage response coordinated by phospho-NBS1 and phospho-CHK1. E1A expression alone fails to induce FANCD2 monoubiquitination, indicating that a productive viral infection and/or replication is required for FANC pathway activation. Our data indicate that Ad5 infection induces FANCD2 activation to promote its own replication. Specifically, we show that FANCD2 is involved in the recombination process that accompanies viral DNA replication. This study provides evidence of a DNA damage-independent function of the FANC pathway and identifies a cellular system involved in Ad5 recombination.

The DeltaNp63 proteins are key allies of BRCA1 in the prevention of basal-like breast cancer.

Little is known about the origin of basal-like breast cancers, an aggressive disease that is highly similar to BRCA1-mutant breast cancers. p63 family proteins that are structurally related to the p53 suppressor protein are known to function in stem cell regulation and stratified epithelia development in multiple tissues, and p63 expression may be a marker of basal-like breast cancers. Here we report that ΔNp63 isoforms of p63 are transcriptional targets for positive regulation by BRCA1. Our analyses of breast cancer tissue microarrays and BRCA1-modulated breast cancer cell lines do not support earlier reports that p63 is a marker of basal-like or BRCA1 mutant cancers. Nevertheless, we found that BRCA1 interacts with the specific p63 isoform ΔNp63γ along with transcription factor isoforms AP-2α and AP-2γ. BRCA1 required ΔNp63γ and AP-2γ to localize to an intronic enhancer region within the p63 gene to upregulate transcription of the ΔNp63 isoforms. In mammary stem/progenitor cells, siRNA-mediated knockdown of ΔNp63 expression resulted in genomic instability, increased cell proliferation, loss of DNA damage checkpoint control, and impaired growth control. Together, our findings establish that transcriptional upregulation of ΔNp63 proteins is critical for BRCA1 suppressor function and that defects in BRCA1-ΔNp63 signaling are key events in the pathogenesis of basal-like breast cancer.

Nek1 kinase functions in DNA damage response and checkpoint control through a pathway independent of ATM and ATR

Never-in-mitosis A related protein kinase 1 (Nek1) is involved early in a DNA damage sensing/repair pathway. We have previously shown that cells without functional Nek1 fail to activate the more distal kinases Chk1 and Chk2 and fail to arrest properly at G1/S or M-phase checkpoints in response to DNA damage. As a consequence, foci of damaged DNA in Nek1 null cells persist long after the instigating insult, and Nek1 null cells develop unstable chromosomes at a rate much higher than identically cultured wild type cells. Here we show that Nek1 functions independently of canonical DNA damage responses requiring the PI3 kinase-like proteins ATM and ATR. Chemical inhibitors of ATM/ATR or mutation of the genes that encode them fail to alter the kinase activity of Nek1 or its localization to nuclear foci of DNA damage. Moreover ATM and ATR activities, including the localization of the proteins to DNA damage sites and phosphorylation of early DNA damage response substrates, are intact in Nek1 -/- murine cells and in human cells with Nek1 expression silenced by siRNA. Our results demonstrate that Nek1 is important for proper checkpoint control and characterize for the first time a DNA damage response that does not directly involve one of the known upstream mediator kinases, ATM or ATR.

Human Papillomavirus 16 E7 Oncoprotein Attenuates DNA Damage Checkpoint Control by Increasing the Proteolytic Turnover of Claspin

The human papillomavirus (HPV) 16 E7 oncoprotein has been reported previously to stimulate DNA damage and to activate host cell DNA damage checkpoints. How HPV-16 E7 maintains proliferation despite activated DNA damage checkpoints is incompletely understood. Here, we provide evidence that cells expressing the HPV-16 E7 oncoprotein can enter mitosis in the presence of DNA damage. We show that this activity of HPV-16 E7 involves attenuation of DNA damage checkpoint control by accelerating the proteolytic turnover of claspin. Claspin mediates the activation of CHK1 by ATR in response to replication stress, and its degradation plays a critical role in DNA damage checkpoint recovery. Expression of a nondegradable mutant of claspin was shown to inhibit mitotic entry in HPV-16 E7-expressing cells. Multiple components of the SCF(beta-TrCP)-based claspin degradation machinery were found deregulated in the presence of HPV-16 E7, including cullin 1, beta-TrCP, Aurora A, and Polo-like kinase-1 (PLK1). In contrast, no difference in the expression level of the claspin deubiquitinating enzyme USP7 was detected. Levels of Aurora A and PLK1 as well as phosphorylated PLK1 at threonine 210, a prerequisite for DNA damage checkpoint recovery, remained detectable following replication stress in HPV-16 E7-expressing cells but not in control cells. In summary, our results suggest that the HPV-16 E7 oncoprotein alleviates DNA damage checkpoint responses and promotes mitotic entry by accelerating claspin degradation through a mechanism that involves deregulation of components of the SCF(beta-TrCP)-based claspin degradation machinery.