p53-driven replication stress in nucleoli of malignant epithelial ovarian cancer.

Malignancies including ovarian cancer (OvCa) are genetically unstable. Genomic integrity is maintained by tumor suppressor p53 and DNA damage response network, which crosstalk to each other via not well characterized mechanisms. In this work, we characterize features of damage-related signals in cultured epithelial OvCa cells and tumor biopsies. We found that endogenous burden of DNA damage in OvCa tissues were ubiquitously accumulated in high-grade malignancies than lower grade of cancer that cannot be obviously explained by disturbed function of in DNA damage response (DDR). In contrast, CHK1 phosphorylation (CHK1-pS345) marking the checkpoint activation in nucleolar compartments are prevalent in high-grade OvCa, coincident to the elevated DNA damage in nucleoplasm. Generation of CHK1-pS345 requires the presence of p53 protein in addition to the well-known activities of ATM/ATR kinases. Apparently, mutant forms of p53 possess higher activity in triggering CHK1 phosphorylation than wild type, implying a potential role of p53 in maintaining rDNA integrity. Loss of p53 function would cause replication stress in nucleoli. Altogether, our study reveals endogenous nucleoli stress in OvCa that is coupled to perturbed function of p53 in DNA repair.

Implication of Ataxia-Telangiectasia-mutated kinase in epithelium-mesenchyme transition.

Impairment of genome instability drives the development of cancer by disrupting anti-cancer barriers. Upon genotoxic insults, DNA damage responsive factors, notably ATM kinase, is crucial to protect genomic integrity while promoting cell death. Meanwhile, cytotoxic therapy-inducing DNA lesions is double-edged sword by causing cancer metastasis based on animal models and clinical observations. The underlying mechanisms for the procancer effect of cytotoxic therapies are poorly understood. Here, we report that cancer cells subjected to cytotoxic treatments elicit dramatic alteration of gene expression controlling the potential of epithelium-mesenchyme transition (EMT). Resultantly, EMT-dependent cell mobility is potently induced upon DNA damage. This stimulation of EMT is mainly Ataxia-Telangiectasia-mutated (ATM)-dependent, as the chemical inhibitor specifically inhibiting ATM kinase activity can suppress the EMT gene expression and thus cell mobility. At last, we show that cancer cells with ATM activation display increased metastatic potential in ovarian cancer tissues. Taken together, we reveal a novel role of ATM in promoting metastatic potential of cancer cells by favoring EMT gene expression.

The Antiresection Activity of the X Protein Encoded by Hepatitis Virus B.

Chronic infection of hepatitis B virus (HBV) is associated with an increased incidence of hepatocellular carcinoma (HCC). HBV encodes an oncoprotein, hepatitis B x protein (HBx), that is crucial for viral replication and interferes with multiple cellular activities including gene expression, histone modifications, and genomic stability. To date, it remains unclear how disruption of these activities contributes to hepatocarcinogenesis. Here, we report that HBV exhibits antiresection activity by disrupting DNA end resection, thus impairing the initial steps of homologous recombination (HR). This antiresection activity occurs in primary human hepatocytes undergoing a natural viral infection-replication cycle as well as in cells with integrated HBV genomes. Among the seven HBV-encoded proteins, we identified HBx as the sole viral factor that inhibits resection. By disrupting an evolutionarily conserved Cullin4A-damage-specific DNA binding protein 1-RING type of E3 ligase, CRL4WDR70 , through its H-box, we show that HBx inhibits H2B monoubiquitylation at lysine 120 at double-strand breaks, thus reducing the efficiency of long-range resection. We further show that directly impairing H2B monoubiquitylation elicited tumorigenesis upon engraftment of deficient cells in athymic mice, confirming that the impairment of CRL4WDR70 function by HBx is sufficient to promote carcinogenesis. Finally, we demonstrate that lack of H2B monoubiquitylation is manifest in human HBV-associated HCC when compared with HBV-free HCC, implying corresponding defects of epigenetic regulation and end resection. Conclusion: The antiresection activity of HBx induces an HR defect and genomic instability and contributes to tumorigenesis of host hepatocytes.

Cell type-specific genotoxicity in estrogen-exposed ovarian and fallopian epithelium.

BACKGROUND: Loss of the genomic stability jeopardize genome stability and promote malignancies. A fraction of ovarian cancer (OvCa) arises from pathological mutations of DNA repair genes that result in highly mutagenic genomes. However, it remains elusive why the ovarian epithelial cells are particularly susceptible to the malfunction of genome surveillance system. METHODS: To explore the genotoxic responses in the unique context of microenvironment for ovarian epithelium that is periodically exposed to high-level steroid hormones, we examined estrogen-induced DNA damage by immunofluorescence in OvCa cell lines, animal and human samples. RESULTS: We found that OvCa cells are burdened with high levels of endogenous DNA damage that is not correlated with genomic replication. The elevation of damage burden is attributable to the excessive concentration of bioactive estrogen instead of its chemomimetic derivative (tamoxifen). Induction of DNA lesions by estrogen is dependent on the expression of hormone receptors, and occurs in G1 and non-G1 phases of cell cycle. Moreover, depletion of homologous recombination (HR) genes (BRCA1 and BRCA2) exacerbated the genotoxicity of estrogen, highlighting the role of HR to counteract hormone-induced genome instability. Finally, the estrogen-induced DNA damage was reproduced in the epithelial compartments of both ovarian and fallopian tubes. CONCLUSIONS: Taken together, our study disclose that estrogen-induced genotoxicity and HR deficiency perturb the genome stability of ovarian and fallopian epithelial cells, representing microenvironmental and genetic risk factors, respectively.

Active DNA end processing in micronuclei of ovarian cancer cells.

BACKGROUND: Ovarian cancer is one of the most deadly gynecological malignancies and inclined to recurrence and drug resistance. Previous studies showed that the tumorigenesis of ovarian cancers and their major histotypes are associated with genomic instability caused by defined sets of pathogenic mutations. In contrast, the mechanism that influences the development of drug resistance and disease recurrence is not well elucidated. Solid tumors are prone to chromosomal instability (CIN) and micronuclei formation (MN). Although MN is traditionally regarded as the outcome of genomic instability, recent investigation on its origin and final consequences reveal that the abnormal DNA metabolism in MN is a driver force for some types of catastrophic genomic rearrangements, accelerating dramatic genetic variation of cancer cells. METHODS: We used Indirect Immunofluorescent staining to visualize micronuclei and activation of DNA repair factors in ovarian cancer cell lines and biopsies. RESULTS: We show that ovarian cancer cells are disposed to form micronuclei upon genotoxic insults. Double strand DNA breaks (DSBs)-triggered insurgence of micronuclei is associated with unrepaired chromosomes passing through mitosis. According to their morphology and DNA staining, micronuclei compartments are divided into early and late stages that can be further characterized by differential staining of γH2AX and 53BP1. We also show that MN compartments do not halt controlled DNA metabolism as sequestered nuclear repair factors are enriched at DNA breaks in MN compartments and efficiently process DNA ends to generate single-stranded DNA (ssDNA) structures. Interestingly, unknown factors are required for DNA end processing in MN in addition to the nuclear resection machinery. Finally, these hallmarks of micronuclei evolution depicted in cell culture were recapitulated in different stages of ovarian cancer biopsies. CONCLUSIONS: In aggregate, our findings demonstrate that ovarian cancer cells are inclined to form micronuclei that undergo robust DNA metabolism and generate ssDNA structures, potentially destabilizing genomic structures and triggering genetic variation.

[DNA Repair Function and Mutation of an H2B Monoubiquitination Factor WDR70 in Ovarian Cancer].

OBJECTIVE: To investigate the roles of enzyme DCAF proteinDNA damagebinding protein 1 (DDB1)/cullin4 (CRL4) complex family members CRL4WD40 repeat domain protein 70 (WDR70) in DNA repair process and its mutation in ovarian cancer. METHODS: Immunofluorescent assay was employed to measure H2AX (γH2AX) and phosphorylated replication protein A2 (RPA32) formed in siDDB1 or siWDR70 ovarian cancer cells after the treatments of chemical medicine and radioactive threapy. 5Brdu immunohistochemical staining was used to explore the function of WDR70 in DNA replication. The expressions of WDR70 and histone protein H2B monoubiquitination (uH2B) was measured by immunohistochemistry,the function of DNA repair,expression and mutations of CRL4 in ovarian cancer were detected by semiquantitative PCR and DNA sequencing. RESULTS: Immunofluorescent assay indicated that distinct subunits of CRL4 played different roles in checkpoint activation and H2Bmonoubiquitinationdepedendent homologous recombination,while the scaffold subunit DDB1 participated in both processes,WDR70 was only required for DNA end resection,chromatin loading of RPA32 and HR. The dose of WDR70 was not effect on DNA replication. Ovarian cancer had different expression of WDR70 and uH2B compared with normal tissue,transcripts of WDR70 was diminished or truncated in 50% of ovarian cancer,which corresponded to multiple mutations. CONCLUSION: CRL4 ubiquitin ligase plays multiple roles in DNA repair and is critical for genome stability. It may be an potential anticancer barrier against ovarian malignancies.

[HPV E7 Function in DNA Damage Response of Cervical Intraepithelial Neoplasia.]

OBJECTIVES: To determine the function of human papillomavirus (HPV) E7 in DNA damage response of cervical intraepithelial neoplasia (CIN) 3 cells. METHODS: Samples of CIN 3 and child foreskin tissues were collected,with pathologically confirmed HPV positive and negative,respectively.Collagenase A was used for digesting tissues prior to primary culture.The HPV negative cells were infected with lentivirus E7 and pLV.Proteins(53BP1,NBS1,BRCA1 and RPA32) responsive to DNA double break damages were detected by indirect immunofluorescent staining after 0-8 h treatment with X-ray (2 or 5 Gy). RESULTS: After treatment with 2 or 5 Gy X-ray,53BP1,NBS1,BRCA1 and RPA32 foci in HPV+ cells increased compared with HPV- cells (P<0.05).Less 53BP1,RPA32,BRCA1 and NBS1 foci positive cells (foci>5) were found in E7 infected cells than in pLV infected cells(P<0.05).Both of them reached the peak at 6 h (2 Gy) or 4 h (5 Gy). CONCLUSIONS: We have successfully established a model to detect the function of HPV E7 in DNA damage response using primary culture of CIN fibroblasts.With the progression of CIN,HPV E7 can inhibit DNA double break repair.

[Employing DNA Damage Response Inhibitors to Enhance Chemosensitivity of Ovarian Carcinoma Cells].

OBJECTIVE: To assess the sensitisation effects of DDR inhibitors combined with conventional chemotherapeutics agents (cisplatin et al) in a drug-resistant ovarian cancer cell line(OVCAR-8). METHODS: Inhibitors of DDR regulators with cisplatin were applied to challenge OVCAR-8, and evaluated the DNA damage response (DDR) and cytotoxic effects of different combination of chemicals. Inhibition of proliferation to OVCAR-8 of different drugs was evaluated by MTT assay. The activation of phosphorylation of histone family 2A variant (yH2AX) and p53 binding protein 1 (53BP1) in OVCAR-8 were evaluated by immunofluorescence to observe their ability of recruitment and forming foci at DNA damage site. RESULTS: We observed that combined treatment of ataxia-telangiectasia mutated (ATM)/ATM and Rad 3-related (ATR) inhibitor and cisplatin can suppress the activation of damage repair mechanisms and weakened the proliferative activity of OVCAR-8 cells (P<0. 01) ; ATR pathway was suppressed and the signal of γH2AX weakened and cell survival rate significantly reduced when combination therapy of HU and Wortmannin (P < 0.05); poly ADP-ribose polymerase (PARP) inhibitor could not enhance chemosensitivity in OVCAR-8 cells when combined with cisplatin (P > 0.05). CONCLUSION: We substantiated that appropriate inhibitors of DNA damage response may have a potential to improve the anti-tumor effect of conventional chemotherapy drugs and prevent drug resistances.

[Functional Analysis of DNA Damage Repair Factor WDR70 and Its Mutation in Ovarian Cancer].

OBJECTIVES: To analyze the cellular function of the newly discovered DNA damage repair factor WDR70, and investigate the mutation in ovarian cancer to verify if function loss of the WDR70gene was associated with ovarian cancer. METHODS: The WDR70 gene was silenced by using siRNA technique or overexpressed its wild and mutation type by with lentivirus and plasmid in hunman cells. The subcellular localization and biochemical function of WDR70 was analyzes by indirect immunofluorescence and immunoblotting. The expression level of WDR70 and the mutations of its cDNA was checked with RT-PCR sequencing for 1 normal ovarian tissue and 16 ovarian cancer specimen. RESULTS: We found gene silencing of WDR70 or overexpression of WDR70 mutation type disrupts the phosphorylation level of homologous recombination functional protein RPA32 and the ability of recruitment at DNA damage site of recombinase RAD51, the loss of function of WDR70 also causes the elevation of the chromosome breakage in metaphase. Meanwhile, we also noticed that the existence of multiple mutations in genomic WDR70 in ovarian cancer specimen. CONCLUSIONS: Our results defined that in vitro system, WDR70 is a DNA damage repair gene, silencing of WDR70 or overexpression of WDR70 mutation type disrupts homologous recombination and chromosomal instability; the frequent mutations of WDR70 gene in genome of ovarian cancer specimens could also lead to DNA repair defeat and gene instability. Consequently WDR70 gene could represent an anti-cancer mechanism for ovarian cancer.

HBx affects CUL4-DDB1 function in both positive and negative manners.

Hepatitis B virus (HBV) infection is a major public health problem by affecting 350 million people worldwide. The mechanisms that regulate HBV gene expression and viral replication remain poorly understood. HBx is known as the central regulator for HBV replication and is associated with the CUL4-DDB1 ubiquitin ligase through H-box motif. Here, we show that blocking the activity of DDB1 by RNA interfering inhibited viral production and gene expression of HBV, and direct association of HBx with DDB1 promoted viral activities, indicating that DDB1 function is required for viral production. On the other hand, HBx interfered with DDB1-dependent polyubiquitination of PRMT1, arginine methyltransferase 1, suggesting that HBx can also block the function of a subset of CUL4-DDB1 E3 ligases. Thus, we conclude that HBx regulates the function of DDB1 in both positive and negative manners in the context of distinct CUL4-DDB1 complexes and plays different roles in HBV replication cycle.

Human papillomavirus E7 protein induces homologous recombination defects and PARPi sensitivity.

PURPOSE: Cervical cancer is a common gynecological malignancy, pathologically associated with persistent infection of high-risk types of human papillomavirus (HPV). Previous studies revealed that HPV-positive cervical cancer displays genomic instability; however, the underlying mechanism is not fully understood. METHODS: To investigate if DNA damage responses are aggravated in precancerous lesions of HPV-positive cervical epithelium, cervical tissues were biopsied and cryosectioned, and subjected to immunofluorescent staining. Cloned HA-tagged E6 and E7 genes of HPV16 subtype were transfected into HEK293T or C33A cells, and indirect immunofluorescent staining was applied to reveal the competency of double strand break (DSB) repair. To test the synthetic lethality of E7-indued HRD and PARP inhibitor (PARPi), we expressed E7 in C33A cells in the presence or absence of olaparib, and evaluated cell viability by colony formation. RESULTS: In precancerous lesions, endogenous DNA lesions were elevated along with the severity of CIN grade. Expressing high-risk viral factor (E7) in HPV-negative cervical cells did not impair checkpoint activation upon genotoxic insults, but affected the potential of DSB repair, leading to homologous recombination deficiency (HRD). Based on this HPV-induced genomic instability, the viability of E7-expressing cells was reduced upon exposure to PARPi in comparison with control cells. CONCLUSION: In aggregate, our findings demonstrate that HPV-E7 is a potential driver for genome instability and provides a new angle to understand its role in cancer development. The viral HRD could be employed to target HPV-positive cervical cancer via synthetic lethality.

Whole transcriptome sequencing reveals genes involved in plastid/chloroplast division and development are regulated by the HP1/DDB1 at an early stage of tomato fruit development.

The phenotype of tomato high pigment-1 (hp1) mutant is characterized by overproduction of pigments including chlorophyll and carotenoids during fruit development and ripening. Although the increased plastid compartment size has been thought to largely attribute to the enhanced pigmentation, the molecular aspects of how the HP1/DDB1 gene manipulates plastid biogenesis and development are largely unknown. In the present study, we compared transcriptome profiles of immature fruit pericarp tissue between tomato cv. Ailsa Craig (WT) and its isogenic hp1 mutant. Over 20 million sequence reads, representing > 1.6 Gb sequence data per sample, were generated and assembled into 21,972 and 22,167 gene models in WT and hp1, respectively, accounting for over 60 % official gene models in both samples. Subsequent analyses revealed that 8,322 and 7,989 alternative splicing events, 8833 or 8510 extended 5'-UTRs, 8,263 or 8,939 extended 3'-UTRs, and 1,136 and 1,133 novel transcripts, exist in WT and hp1, respectively. Significant differences in expression level of 880 genes were detected between the WT and hp1, many of which are involved in signaling transduction, transcription regulation and biotic and abiotic stresses response. Distinctly, RNA-seq datasets, quantitative RT-PCR analyses demonstrate that, in hp1 mutant pericarp tissue at early developmental stage, an apparent expression alteration was found in several regulators directly involved in plastid division and development. These results provide a useful reference for a more accurate and more detailed characterization of the molecular process in the development and pigmentation of tomato fruits.

Hepatitis B virus infection disrupts homologous recombination in hepatocellular carcinoma by stabilizing resection inhibitor ADRM1.

Many cancers harbor homologous recombination defects (HRDs). A HRD is a therapeutic target that is being successfully utilized in treatment of breast/ovarian cancer via synthetic lethality. However, canonical HRD caused by BRCAness mutations do not prevail in liver cancer. Here we report a subtype of HRD caused by the perturbation of a proteasome variant (CDW19S) in hepatitis B virus-bearing (HBV-bearing) cells. This amalgamate protein complex contained the 19S proteasome decorated with CRL4WDR70 ubiquitin ligase, and assembled at broken chromatin in a PSMD4Rpn10- and ATM-MDC1-RNF8-dependent manner. CDW19S promoted DNA end processing via segregated modules that promote nuclease activities of MRE11 and EXO1. Contrarily, a proteasomal component, ADRM1Rpn13, inhibited resection and was removed by CRL4WDR70-catalyzed ubiquitination upon commitment of extensive resection. HBx interfered with ADRM1Rpn13 degradation, leading to the imposition of ADRM1Rpn13-dependent resection barrier and consequent viral HRD subtype distinguishable from that caused by BRCA1 defect. Finally, we demonstrated that viral HRD in HBV-associated hepatocellular carcinoma can be exploited to restrict tumor progression. Our work clarifies the underlying mechanism of a virus-induced HRD subtype.

Synthetic lethality between TP53 and ENDOD1.

The atypical nuclease ENDOD1 functions with cGAS-STING in innate immunity. Here we identify a previously uncharacterized ENDOD1 function in DNA repair. ENDOD1 is enriched in the nucleus following H2O2 treatment and ENDOD1-/- cells show increased PARP chromatin-association. Loss of ENDOD1 function is synthetic lethal with homologous recombination defects, with affected cells accumulating DNA double strand breaks. Remarkably, we also uncover an additional synthetic lethality between ENDOD1 and p53. ENDOD1 depletion in TP53 mutated tumour cells, or p53 depletion in ENDOD1-/- cells, results in rapid single stranded DNA accumulation and cell death. Because TP53 is mutated in ~50% of tumours, ENDOD1 has potential as a wide-spectrum target for synthetic lethal treatments. To support this we demonstrate that systemic knockdown of mouse EndoD1 is well tolerated and whole-animal siRNA against human ENDOD1 restrains TP53 mutated tumour progression in xenograft models. These data identify ENDOD1 as a potential cancer-specific target for SL drug discovery.

Wdr70 regulates histone modification and genomic maintenance in fission yeast.

Eukaryotic genomes are packaged into highly condensed chromatin and this repressive chromatin barrier can be overcome by altering the chromatin structure via histone modification enzymes. Here, we report Wdr70 in Schizosaccharomyces pombe (spWdr70) plays important roles in multiple cellular processes including cell cycle progression, chromatin structure and DNA repair. Depletion of Wdr70 gene causes cell cycle delay, hypersensitivity to DNA damage reagents and quick phenotypic changes. Moreover, we observed strong genetic interaction between Wdr70 and genes regulating checkpoint and homologous recombination (HR), pinpointing the function of Wdr70 to DNA end resection. Finally, we show that the function of Wdr70 could be attributed to monoubiquitination of histone H2B (uH2B) in the vicinity of DNA double strand breaks (DSBs). Taken together, our data reveal that Wdr70 and H2B monoubiquitination-dependent chromatin modulation is required for chromatin homeostasis and genetic stability.

CRL4(Wdr70) regulates H2B monoubiquitination and facilitates Exo1-dependent resection.

Double-strand breaks repaired by homologous recombination (HR) are first resected to form single-stranded DNA, which binds replication protein A (RPA). RPA attracts mediators that load the Rad51 filament to promote strand invasion, the defining feature of HR. How the resection machinery navigates nucleosome-packaged DNA is poorly understood. Here we report that in Schizosaccharomyces pombe a conserved DDB1-CUL4-associated factor (DCAF), Wdr70, is recruited to DSBs as part of the Cullin4-DDB1 ubiquitin ligase (CRL4(Wdr70)) and stimulates distal H2B lysine 119 mono-ubiquitination (uH2B). Wdr70 deletion, or uH2B loss, results in increased loading of the checkpoint adaptor and resection inhibitor Crb2(53BP1), decreased Exo1 association and delayed resection. Wdr70 is dispensable for resection upon Crb2(53BP1) loss, or when the Set9 methyltransferase that creates docking sites for Crb2 is deleted. Finally, we establish that this histone regulatory cascade similarly controls DSB resection in human cells.

An unusual posttranscriptional processing in two betaine aldehyde dehydrogenase loci of cereal crops directed by short, direct repeats in response to stress conditions.

Various abilities to synthesize and accumulate glycine betaine (GB) are crucial for angiosperms to develop salt and drought tolerances. In higher plants, GB is synthesized by a two-step oxidation of choline via an intermediate form of betaine aldehyde, and catalyzed by choline monooxygenase and betaine aldehyde dehydrogenase (BADH). In this study, numerous truncated and/or recombinant transcripts of two BADH homologs resulting from an unusual posttranscriptional processing were detected in rice (Oryza sativa) and other cereal crops, including maize (Zea mays), wheat (Triticum aestivum), and barley (Hordeum vulgare). The observed events took place at the 5' exonic region, and led to the insertion of exogenous gene sequences and a variety of deletions that resulted in the removal of translation initiation codon, loss of functional domain, and frame-shifts with premature termination by introducing stop codon. By contrast, the BADH transcripts from dicotyledonous species, such as spinach (Spinacia oleracea), Arabidopsis (Arabidopsis thaliana), and tomato (Solanum lycopersicum), had correctly processed mRNA. This suggests the differentiation of posttranscriptional processing in BADH genes potentially contributes to the variation of GB-synthesizing capacities among various plant species. In addition, comprehensive sequence analyses demonstrated that extensive sequence similarities (named as short, direct repeats) are of paired presence surrounding the junctions of both the deletion and/or insertion sites in the unusual BADH transcripts. The site selection for the deletion/insertion was altered in response to the stress conditions. This indicates that the sequence elements of short, direct repeats are probably required for the recognition of the deletion/insertion sites.