Long noncoding RNA TUG1 promotes cisplatin resistance in ovarian cancer via upregulation of DNA polymerase eta.

Chemoresistance is a major cause of high mortality and poor survival in patients with ovarian cancer (OVCA). Understanding the mechanisms of chemoresistance is urgently required to develop effective therapeutic approaches to OVCA. Here, we show that expression of the long noncoding RNA, taurine upregulated gene 1 (TUG1), is markedly upregulated in samples from OVCA patients who developed resistance to primary platinum-based therapy. Depletion of TUG1 increased sensitivity to cisplatin in the OVCA cell lines, SKOV3 and KURAMOCHI. Combination therapy of cisplatin with antisense oligonucleotides targeting TUG1 coupled with a drug delivery system effectively relieved the tumor burden in xenograft mouse models. Mechanistically, TUG1 acts as a competing endogenous RNA by downregulating miR-4687-3p and miR-6088, both of which target DNA polymerase eta (POLH), an enzyme required for translesion DNA synthesis. Overexpression of POLH reversed the effect of TUG1 depletion on cisplatin-induced cytotoxicity. Our data suggest that TUG1 upregulation allows OVCA to tolerate DNA damage via upregulation of POLH; this provides a strong rationale for targeting TUG1 to overcome cisplatin resistance in OVCA.

Successful i-GONAD in Mice at Early Zygote Stage through In Vivo Electroporation Three Min after Intraoviductal Instillation of CRISPR-Ribonucleoprotein.

Improved genome editing via oviductal nucleic acids delivery (i-GONAD) is a new technology enabling in situ genome editing of mammalian zygotes exiting the oviductal lumen, which is now available in mice, rats, and hamsters. In this method, CRISPR/Cas9 genome-editing reagents are delivered directly to the oviducts of pregnant animals (corresponding to late zygote stage). After intraoviductal instillation, electric shock to the entire oviduct was provided with a specialized electroporation (EP) device to drive the genome editing reagents into the zygotes present in the oviductal lumen. i-GONAD toward early zygotes has been recognized as difficult, because they are tightly surrounded by a cumulus cell layer, which often hampers effective transfer of nucleic acids to zygotes. However, in vivo EP three min after intraoviductal instillation of the genome-editing reagents enabled genome editing of early zygotes with an efficiency of 70%, which was in contrast with the rate of 18% when in vivo EP was performed immediately after intraoviductal instillation at Day 0.5 of pregnancy (corresponding to 13:00-13:30 p.m. on the day when vaginal plug was recognized after natural mating). We also found that addition of hyaluronidase, an enzyme capable of removing cumulus cells from a zygote, slightly enhanced the efficiency of genome editing in early zygotes. These findings suggest that cumulus cells surrounding a zygote can be a barrier for efficient generation of genome-edited mouse embryos and indicate that a three-minute interval before in vivo EP is effective for achieving i-GONAD-mediated genome editing at the early zygote stage. These results are particularly beneficial for researchers who want to perform genome editing experiments targeting early zygotes.

Rare-earth ions as antibacterial agents for woven wool fabric.

Woven fabrics were bestowed with antibacterial property by the simple adsorption of rare-earth metal ions, and the underlying mechanism was investigated using electron spin resonance (ESR) spectroscopy. The adsorption of Ce3+ ions on wool, silk, and cotton fabrics resulted in significant inhibition of Staphylococcus aureus (a gram-positive bacterium), with maximum antibacterial activities (viable bacterial count compared to the reference) of 4.7, 5.8, and 5.2, respectively. Even after 50 wash cycles, the values remained at 3.9, 2.9, and 4.8, respectively. The adsorption of La3+ and Gd3+ ions on wool fabrics also resulted in antibacterial activities of 5.8 and 5.9, respectively. In addition, wool adsorbed with Ce3+ exhibits a satisfactory antibacterial activity of 6.2 against Escherichia coli (a gram-negative bacterium). Such bacterial inhibition is attributed to Fenton reactions between the adsorbed rare-earth ions and hydrogen peroxide (H2O2) produced during bacterial metabolism, as determined from the ESR spectra collected using the spin trap method in the presence of H2O2. The safety of cerium nitrate was also investigated, and no significant issues arose, indicating that it was a safe antibacterial agent. This facile method of imparting antibacterial properties to natural fabrics may be useful for preventing infections in humans. Supplementary Information: The online version contains supplementary material available at 10.1007/s11696-021-01999-9.

Modification of improved-genome editing via oviductal nucleic acids delivery (i-GONAD)-mediated knock-in in rats.

BACKGROUND: Improved genome-editing via oviductal nucleic acids delivery (i-GONAD) is a new technology that facilitates in situ genome-editing of mammalian zygotes exiting the oviductal lumen. The i-GONAD technology has been developed for use in mice, rats, and hamsters; however, oligonucleotide (ODN)-based knock-in (KI) is more inefficient in rats than mice. To improve the efficiency of i-GONAD in rats we examined KI efficiency using three guide RNAs (gRNA), crRNA1, crRNA2 and crRNA3. These gRNAs recognize different portions of the target locus, but also overlap each other in the target locus. We also examined the effects of commercially available KI -enhancing drugs (including SCR7, L755,507, RS-1, and HDR enhancer) on i-GONAD-mediated KI efficiency. RESULTS: The KI efficiency in rat fetuses generated after i-GONAD with crRNA2 and single-stranded ODN was significantly higher (24%) than crRNA1 (5%; p < 0.05) or crRNA3 (0%; p < 0.01). The KI efficiency of i-GONAD with triple gRNAs was 11%. These findings suggest that KI efficiency largely depends on the type of gRNA used. Furthermore, the KI efficiency drugs, SCR7, L755,507 and HDR enhancer, all of which are known to enhance KI efficiency, increased KI efficiency using the i-GONAD with crRNA1 protocol. In contrast, only L755,507 (15 µM) increased KI efficiency using the i-GONAD with crRNA2 protocol. None of them were significantly different. CONCLUSIONS: We attempted to improve the KI efficiency of i-GONAD in rats. We demonstrated that the choice of gRNA is important for determining KI efficiency and insertion and deletion rates. Some drugs (e.g. SCR7, L755,507 and HDR enhancer) that are known to increase KI efficiency in culture cells were found to be effective in i-GONAD in rats, but their effects were limited.

Development of a protein-based system for transient epigenetic repression of immune checkpoint molecule and enhancement of antitumour activity of natural killer cells.

BACKGROUND: Immune checkpoint blockade (ICB) therapy improved the prognosis of cancer patients, but general administration of ICBs occasionally induces side effects that include immune-related adverse events and tumour hyper-progression. Here, we established a protein-based system, by which endogenous expression of IC molecule in natural killer (NK) cells was transiently repressed on enhancement of their antitumour activity. METHODS: A protein-based genome modulator (GM) system is composed of a transcription activator-like effector (TALE), DNA methyltransferase and a newly identified potent cell-penetrating peptide with nuclear-trafficking property named NTP. TALE was designed to target the promoter region of the programmed cell death-1 (PD-1) gene. After culturing human NK cells in the presence of NTP-GM protein, we examined endogenous PD-1 expression and antitumour activity of the treated cells. RESULTS: NTP-GM protein efficiently downregulated PD-1 expression in NK cells with increased CpG DNA methylation in the promoter region. The antitumour activity of the treated NK cells was enhanced, and repeated intraperitoneal administrations of the treated NK cells attenuated tumour growth of programmed death-ligand 1-positive tumour cells in vivo. CONCLUSIONS: Because the incorporated NTP-GM protein was quickly degraded and negligible in the administered NK cells, the NTP-GM system could be an alternative option of an ICB without side effects.

Structural alteration of DNA induced by viral protein R of HIV-1 triggers the DNA damage response.

BACKGROUND: Viral protein R (Vpr) is an accessory protein of HIV-1, which is potentially involved in the infection of macrophages and the induction of the ataxia-telangiectasia and Rad3-related protein (ATR)-mediated DNA damage response (DDR). It was recently proposed that the SLX4 complex of structure-specific endonuclease is involved in Vpr-induced DDR, which implies that aberrant DNA structures are responsible for this phenomenon. However, the mechanism by which Vpr alters the DNA structures remains unclear. RESULTS: We found that Vpr unwinds double-stranded DNA (dsDNA) and invokes the loading of RPA70, which is a single-stranded DNA-binding subunit of RPA that activates the ATR-dependent DDR. We demonstrated that Vpr influenced RPA70 to accumulate in the corresponding region utilizing the LacO/LacR system, in which Vpr can be tethered to the LacO locus. Interestingly, RPA70 recruitment required chromatin remodelling via Vpr-mediated ubiquitination of histone H2B. On the contrary, Q65R mutant of Vpr, which lacks ubiquitination activity, was deficient in both chromatin remodelling and RPA70 loading on to the chromatin. Moreover, Vpr-induced unwinding of dsDNA coincidently resulted in the accumulation of negatively supercoiled DNA and covalent complexes of topoisomerase 1 and DNA, which caused DNA double-strand breaks (DSBs) and DSB-directed integration of proviral DNA. Lastly, we noted the dependence of Vpr-promoted HIV-1 infection in resting macrophages on topoisomerase 1. CONCLUSIONS: The findings of this study indicate that Vpr-induced structural alteration of DNA is a primary event that triggers both DDR and DSB, which ultimately contributes to HIV-1 infection.

Monocyte Chemoattractant Protein-1 (MCP-1) as a Potential Therapeutic Target and a Noninvasive Biomarker of Liver Fibrosis Associated With Transient Myeloproliferative Disorder in Down Syndrome.

Liver fibrosis is one of the common complications of transient myeloproliferative disorder (TMD) in Down syndrome (DS), but the exact molecular pathogenesis is largely unknown. We herein report a neonate of DS with liver fibrosis associated with TMD, in which we performed the serial profibrogenic cytokines analyses. We found the active monocyte chemoattractant protein-1 expression in the affected liver tissue and also found that both serum and urinary monocyte chemoattractant protein-1 concentrations are noninvasive biomarkers of liver fibrosis. We also showed a prospective of the future anticytokine therapy with herbal medicine for the liver fibrosis associated with TMD in DS.

Viral protein R of HIV type-1 induces retrotransposition and upregulates glutamate synthesis by the signal transducer and activator of transcription 1 signaling pathway.

Viral protein R (Vpr) of HIV-1 plays an important role in viral replication in macrophages. Various lines of evidence suggest that expression of Vpr in macrophages causes immunopathogenesis; however, the underlying mechanism is not yet fully understood. In this study, it was shown that recombinant Vpr (rVpr) induces retrotransposition of long interspersed element-1 in RAW264.7, a macrophage-like cell line, and activates reverse transcriptase-dependent immunotoxic cascades including production of IFN-ß and phosphorylation of signal transducer and activator of transcription 1 (STAT1). Knockout experiments based on the CRISPR/Cas9 nickase system further demonstrated that cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) and stimulator of interferon gene (STING) are responsible for IFN-ß production and STAT1 phosphorylation, respectively. Moreover, rVpr was found to increase production of glutaminase C, a regulator of glutamate synthesis, which is also dependent on the cGAS-STING pathway. Taken together with reports that glutaminase C is involved in the pathogenesis of HIV-associated neurocognitive disorder (HAND) and that Vpr is detectable in the cerebrospinal fluid of HIV-1-positive patients, a possible role of Vpr-induced L1-RTP and immunotoxic cascades in the development of HAND is discussed.

Viral protein R of human immunodeficiency virus type-1 induces retrotransposition of long interspersed element-1.

BACKGROUND: Viral protein R (Vpr), a protein of human immunodeficiency virus type-1 (HIV-1) with various biological functions, was shown to be present in the blood of HIV-1-positive patients. However, it remained unclear whether circulating Vpr in patients' blood is biologically active. Here, we examined the activity of blood Vpr using an assay system by which retrotransposition of long interspersed element-1 (L1-RTP) was detected. We also investigated the in vivo effects of recombinant Vpr (rVpr) by administrating it to transgenic mice harboring human L1 as a transgene (hL1-Tg mice). Based on our data, we discuss the involvement of blood Vpr in the clinical symptoms of acquired immunodeficiency syndrome (AIDS). RESULTS: We first discovered that rVpr was active in induction of L1-RTP. Biochemical analyses revealed that rVpr-induced L1-RTP depended on the aryl hydrocarbon receptor, mitogen-activated protein kinases, and CCAAT/enhancer-binding protein ß. By using a sensitive L1-RTP assay system, we showed that 6 of the 15 blood samples from HIV-1 patients examined were positive for induction of L1-RTP. Of note, the L1-RTP-inducing activity was blocked by a monoclonal antibody specific for Vpr. Moreover, L1-RTP was reproducibly induced in various organs, including the kidney, when rVpr was administered to hL1-Tg mice. CONCLUSIONS: Blood Vpr is biologically active, suggesting that its monitoring is worthwhile for clarification of the roles of Vpr in the pathogenesis of AIDS. This is the first report to demonstrate a soluble factor in patients' blood active for L1-RTP activity, and implies the involvement of L1-RTP in the development of human diseases.

Induction of long interspersed nucleotide element-1 (L1) retrotransposition by 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan photoproduct.

Long interspersed nucleotide element-1 (L1) is a retroelement comprising about 17% of the human genome, of which 80-100 copies are competent as mobile elements (retrotransposition: L1-RTP). Although the genetic structures modified during L1-RTP have been clarified, little is known about the cellular signaling cascades involved. Herein we found that 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan photoproduct postulated as a candidate physiological ligand of the aryl hydrocarbon receptor (AhR), induces L1-RTP. Notably, RNA-interference experiments combined with back-transfection of siRNA-resistant cDNAs revealed that the induction of L1-RTP by FICZ is dependent on AhR nuclear translocator-1 (ARNT1), a binding partner of AhR, and the activation of cAMP-responsive element-binding protein. However, our extensive analyses suggested that AhR is not required for L1-RTP. FICZ stimulated the interaction of the L1-encoded open reading frame-1 (ORF1) and ARNT1, and recruited ORF1 to chromatin in a manner dependent on the activation of mitogen-activated protein kinase. Along with our additional observations that the cellular cascades for FICZ-induced L1-RTP were different from those of L1-RTP triggered by DNA damage, we propose that the presence of the cellular machinery of ARNT1 mediates L1-RTP. A possible role of ARNT1-mediated L1-RTP in the adaptation of living organisms to environmental changes is discussed.

Ceftriaxone-Induced Neutropenia Successfully Treated With Alternative ß-Lactam Antibiotics: A Case Report and Review of the Literature.

Ceftriaxone-induced neutropenia is a rare and severe adverse effect of the drug. It usually resolves in one to three weeks following the cessation of ceftriaxone and the administration of granulocyte colony-stimulating factor (G-CSF). After neutrophil recovery, patients are often treated with non-ß-lactam antibiotics instead of ceftriaxone due to the possibility of cross-reactivity associated with ß-lactam allergy. However, in some cases, ß-lactam antibiotics are superior to non-ß-lactam antibiotics. Few cases of the readministration of ß-lactam antibiotics for patients who developed ceftriaxone-induced neutropenia have been reported so far. Moreover, its pathogenesis and management have still not been established. We describe a case of successful readministration of ß-lactam antibiotics for a patient who had developed ceftriaxone-induced neutropenia. A 37-year-old man with a prosthetic aortic valve was admitted to our hospital with a fever. Blood culture on admission revealed methicillin-susceptible Staphylococcus aureus (MSSA) bacteremia, and transesophageal echocardiography (TEE) showed aortic valve vegetation with multiple septic emboli seen on brain CT. We diagnosed MSSA infective endocarditis with central nervous complications. He underwent an operation and was treated with ceftriaxone. On admission day 28, he developed neutropenia (33/µL), and ceftriaxone-induced neutropenia was suspected. Vancomycin was started instead of ceftriaxone, and his neutrophil count recovered within two weeks with the administration of G-CSF. After recovery, on day 40 of admission, ampicillin sodium was administered instead of vancomycin. Although he developed mild eosinophilia, he did not exhibit neutropenia and was discharged with an amoxicillin prescription on day 60 of admission. Our report suggests the possibility that patients who develop ceftriaxone-induced neutropenia can be treated safely with an alternative ß-lactam antibiotic, ampicillin sodium, without causing ß-lactam cross-reactivity of neutropenia.

p130RB2 positively contributes to ATR activation in response to replication stress via the RPA32-ETAA1 axis.

Ataxia-telangiectasia mutated and Rad3-related (ATR) kinase is a crucial regulator of the cell cycle checkpoint and activated in response to DNA replication stress by two independent pathways via RPA32-ETAA1 and TopBP1. However, the precise activation mechanism of ATR by the RPA32-ETAA1 pathway remains unclear. Here, we show that p130RB2, a member of the retinoblastoma protein family, participates in the pathway under hydroxyurea-induced DNA replication stress. p130RB2 binds to ETAA1, but not TopBP1, and depletion of p130RB2 inhibits the RPA32-ETAA1 interaction under replication stress. Moreover, p130RB2 depletion reduces ATR activation accompanied by phosphorylation of its targets RPA32, Chk1, and ATR itself. It also causes improper re-progression of S phase with retaining single-stranded DNA after cancelation of the stress, which leads to an increase in the anaphase bridge phenotype and a decrease in cell survival. Importantly, restoration of p130RB2 rescued the disrupted phenotypes of p130RB2 knockdown cells. These results suggest positive involvement of p130RB2 in the RPA32-ETAA1-ATR axis and proper re-progression of the cell cycle to maintain genome integrity.

Fusobacterium infection facilitates the development of endometriosis through the phenotypic transition of endometrial fibroblasts.

Retrograde menstruation is a widely accepted cause of endometriosis. However, not all women who experience retrograde menstruation develop endometriosis, and the mechanisms underlying these observations are not yet understood. Here, we demonstrated a pathogenic role of Fusobacterium in the formation of ovarian endometriosis. In a cohort of women, 64% of patients with endometriosis but <10% of controls were found to have Fusobacterium infiltration in the endometrium. Immunohistochemical and biochemical analyses revealed that activated transforming growth factor-ß (TGF-ß) signaling resulting from Fusobacterium infection of endometrial cells led to the transition from quiescent fibroblasts to transgelin (TAGLN)-positive myofibroblasts, which gained the ability to proliferate, adhere, and migrate in vitro. Fusobacterium inoculation in a syngeneic mouse model of endometriosis resulted in a marked increase in TAGLN-positive myofibroblasts and increased number and weight of endometriotic lesions. Furthermore, antibiotic treatment largely prevented establishment of endometriosis and reduced the number and weight of established endometriotic lesions in the mouse model. Our data support a mechanism for the pathogenesis of endometriosis via Fusobacterium infection and suggest that eradication of this bacterium could be an approach to treat endometriosis.

TUG1-mediated R-loop resolution at microsatellite loci as a prerequisite for cancer cell proliferation.

Oncogene-induced DNA replication stress (RS) and consequent pathogenic R-loop formation are known to impede S phase progression. Nonetheless, cancer cells continuously proliferate under such high-stressed conditions through incompletely understood mechanisms. Here, we report taurine upregulated gene 1 (TUG1) long noncoding RNA (lncRNA), which is highly expressed in many types of cancers, as an important regulator of intrinsic R-loop in cancer cells. Under RS conditions, TUG1 is rapidly upregulated via activation of the ATR-CHK1 signaling pathway, interacts with RPA and DHX9, and engages in resolving R-loops at certain loci, particularly at the CA repeat microsatellite loci. Depletion of TUG1 leads to overabundant R-loops and enhanced RS, leading to substantial inhibition of tumor growth. Our data reveal a role of TUG1 as molecule important for resolving R-loop accumulation in cancer cells and suggest targeting TUG1 as a potent therapeutic approach for cancer treatment.

Chromatin-remodeling factor BAZ1A/ACF1 targets UV damage sites in an MLL1-dependent manner to facilitate nucleotide excision repair.

Ultraviolet (UV) light irradiation generates pyrimidine dimers on DNA, such as cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts. Such dimers distort the high-order DNA structure and prevent transcription and replication. The nucleotide excision repair (NER) system contributes to resolving this type of DNA lesion. There are two pathways that recognize pyrimidine dimers. One acts on transcribed strands of DNA (transcription-coupled NER), and the other acts on the whole genome (global genome-NER; GG-NER). In the latter case, DNA damage-binding protein 2 (DDB2) senses pyrimidine dimers with several histone modification enzymes. We previously reported that histone acetyltransferase binding to ORC1 (HBO1) interacts with DDB2 and facilitates recruitment of the imitation switch chromatin remodeler at UV-irradiated sites via an unknown methyltransferase. Here, we found that the phosphorylated histone methyltransferase mixed lineage leukemia 1 (MLL1) was maintained at UV-irradiated sites in an HBO1-dependent manner. Furthermore, MLL1 catalyzed histone H3K4 methylation and recruited the chromatin remodeler bromodomain adjacent to zinc finger domain 1A (BAZ1A)/ATP-utilizing chromatin assembly and remodeling factor 1 (ACF1). Depletion of MLL1 suppressed BAZ1A accumulation at UV-irradiated sites and inhibited the removal of CPDs. These data indicate that the DDB2-HBO1-MLL1 axis is essential for the recruitment of BAZ1A to facilitate GG-NER.

Mitotic cells can repair DNA double-strand breaks via a homology-directed pathway.

The choice of repair pathways of DNA double-strand breaks (DSBs) is dependent upon the cell cycle phases. While homologous recombination repair (HRR) is active between the S and G2 phases, its involvement in mitotic DSB repair has not been examined in detail. In the present study, we developed a new reporter assay system to detect homology-directed repair (HDR), a major pathway used for HRR, in combination with an inducible DSB-generation system. As expected, the maximal HDR activity was observed in the late S phase, along with minimal activity in the G1 phase and at the G1/S boundary. Surprisingly, significant HDR activity was observed in M phase, and the repair efficiency was similar to that observed in late S phase. HDR was also confirmed in metaphase cells collected with continuous colcemid exposure. ChIP assays revealed the recruitment of RAD51 to the vicinity of DSBs in M phase. In addition, the ChIP assay for gamma-H2AX and phosphorylated DNA-PKcs indicated that a part of M-phase cells with DSBs could proceed into the next G1 phase. These results provide evidence showing that a portion of mitotic cell DSBs are undoubtedly repaired through action of the HDR repair pathway.

Cancer-Specific Targeting of Taurine-Upregulated Gene 1 Enhances the Effects of Chemotherapy in Pancreatic Cancer.

Overcoming drug resistance is one of the biggest challenges in cancer chemotherapy. In this study, we examine whether targeting the long noncoding RNA taurine upregulated gene 1 (TUG1) could be an effective therapeutic approach to overcome drug resistance in pancreatic ductal adenocarcinoma (PDAC). TUG1 was expressed at significantly higher levels across 197 PDAC tissues compared with normal pancreatic tissues. Overall survival of patients with PDAC who had undergone 5-FU-based chemotherapy was shorter in high TUG1 group than in low TUG1 group. Mechanistically, TUG1 antagonized miR-376b-3p and upregulated dihydropyrimidine dehydrogenase (DPD). TUG1 depletion induced susceptibility to 5-FU in BxPC-3 and PK-9 pancreatic cell lines. Consistently, the cellular concentration of 5-FU was significantly higher under TUG1-depleted conditions. In PDAC xenograft models, intravenous treatment with a cancer-specific drug delivery system (TUG1-DDS) and 5-FU significantly suppressed PDAC tumor growth compared with 5-FU treatment alone. This novel approach using TUG1-DDS in combination with 5-FU may serve as an effective therapeutic option to attenuate DPD activity and meet appropriate 5-FU dosage requirements in targeted PDAC cells, which can reduce the systemic adverse effects of chemotherapy. SIGNIFICANCE: Targeting TUG1 coupled with a cancer-specific drug delivery system effectively modulates 5-FU catabolism in TUG1-overexpressing PDAC cells, thus contributing to a new combinatorial strategy for cancer treatment. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/7/1654/F1.large.jpg.

Homologous recombination but not nucleotide excision repair plays a pivotal role in tolerance of DNA-protein cross-links in mammalian cells.

DNA-protein cross-links (DPCs) are unique among DNA lesions in their unusually bulky nature. The steric hindrance imposed by cross-linked proteins (CLPs) will hamper DNA transactions, such as replication and transcription, posing an enormous threat to cells. In bacteria, DPCs with small CLPs are eliminated by nucleotide excision repair (NER), whereas oversized DPCs are processed exclusively by RecBCD-dependent homologous recombination (HR). Here we have assessed the roles of NER and HR for DPCs in mammalian cells. We show that the upper size limit of CLPs amenable to mammalian NER is relatively small (8-10 kDa) so that NER cannot participate in the repair of chromosomal DPCs in mammalian cells. Moreover, CLPs are not polyubiquitinated and hence are not subjected to proteasomal degradation prior to NER. In contrast, HR constitutes the major pathway in tolerance of DPCs as judged from cell survival and RAD51 and gamma-H2AX nuclear foci formation. Induction of DPCs results in the accumulation of DNA double strand breaks in HR-deficient but not HR-proficient cells, suggesting that fork breakage at the DPC site initiates HR and reactivates the stalled fork. DPCs activate both ATR and ATM damage response pathways, but there is a time lag between two responses. These results highlight the differential involvement of NER in the repair of DPCs in bacterial and mammalian cells and demonstrate the versatile and conserved role of HR in tolerance of DPCs among species.

NBS1 regulates a novel apoptotic pathway through Bax activation.

DNA damage induced apoptosis, along with precise DNA damage repair, is a critical cellular function, and both of these functions are necessary for cancer prevention. The NBS1 protein is known to be a key regulator of DNA damage repair. It acts by forming a complex with Rad50/Mre11 and by activating ATM. We show here that NBS1 regulates a novel p53 independent apoptotic pathway in response to DNA damage. DNA damage induced apoptosis was significantly reduced in NBS1 deficient cells regardless of their p53 status. Experiments using a series of cell lines expressing mutant NBS1 proteins revealed that NBS1 is able to regulate the activation of Bax and Caspase-3 without the FHA, Mre11-binding, or the ATM-interacting domains, whereas the phosphorylation sites of NBS1 were essential for Bax activation. Expression of apoptosis-related transcription factors such as E2F1 and their downstream pro-apoptotic factors were not related to this apoptosis induction. Interestingly, NBS1 regulates a novel Bax activation pathway by disrupting the Ku70-Bax complex which is required for activation of the mitochondrial apoptotic pathway. This dissociation of the Ku70-Bax complex can be mediated by acetylation of Ku70, and NBS1 can function in this process through a protein-protein interaction with Ku70. Thus, NBS1 is a key protein involved in the prevention of carcinogenesis, not only through the precise repair of damaged DNA by homologous recombination (HR) but also by its role in the elimination of inappropriately repaired cells.

Short Communication: A Quantitative System for Monitoring Blood-Circulating Viral Protein R of Human Immunodeficiency Virus-1 Detected a Possible Link with Pathogenic Indices.

We developed a detergent-free enzyme-linked immunosorbent assay (ELISA) for HIV-1 viral protein R (Vpr), an accessory protein of human immunodeficiency virus type-1 (HIV), and detected soluble Vpr in ∼22% of HIV patients who were receiving combination antiretroviral therapy and were free of plasma HIV RNA. Notably, the levels of CD8-positive cell count, soluble intercellular adhesion molecule-1 (sICAM-1), and C-C motif chemokine ligand 2 (CCL2), all of which are markers of chronic inflammation in HIV patients, were higher in Vpr-positive patients than in Vpr-negative patients. Because sICAM1 and CCL2 are associated with an increased risk of HIV-associated neurocognitive disorder, we propose that an established Vpr-ELISA would be useful for monitoring the risk of HIV complications during latent HIV infection.