BAP1 promotes the repair of UV-induced DNA damage via PARP1-mediated recruitment to damage sites and control of activity and stability.

BRCA1-associated protein-1 (BAP1) is a ubiquitin C-terminal hydrolase domain-containing deubiquitinase with tumor suppressor activity. The gene encoding BAP1 is mutated in various human cancers, with particularly high frequency in kidney and skin cancers, and BAP1 is involved in many cancer-related cellular functions, such as DNA repair and genome stability. Although BAP1 stimulates DNA double-strand break repair, whether it functions in nucleotide excision repair (NER) is unknown. Here, we show that BAP1 promotes the repair of ultraviolet (UV)-induced DNA damage via its deubiquitination activity in various cell types, including primary melanocytes. Poly(ADP-ribose) polymerase 1 (PARP1) interacts with and recruits BAP1 to damage sites, with BAP1 recruitment peaking after the DDB2 and XPC damage sensors. BAP1 recruitment also requires histone H2A monoubiquitinated at Lys119, which accumulates at damage sites. PARP1 transiently poly(ADP-ribosyl)ates (PARylates) BAP1 at multiple sites after UV damage and stimulates the deubiquitination activity of BAP1 both intrinsically and via PARylation. PARP1 also promotes BAP1 stability via crosstalk between PARylation and ubiquitination. Many PARylation sites in BAP1 are mutated in various human cancers, among which the glutamic acid (Glu) residue at position 31, with particularly frequent mutation in kidney cancer, plays a critical role in BAP1 stabilization and promotes UV-induced DNA damage repair. Glu31 also participates in reducing the viability of kidney cancer cells. This study therefore reveals that BAP1 functions in the NER pathway and that PARP1 plays a role as a novel factor that regulates BAP1 enzymatic activity, protein stability, and recruitment to damage sites. This activity of BAP1 in NER, along with its cancer cell viability-reducing activity, may account for its tumor suppressor function.

Eukaryotic DNA damage responses: Homologous recombination factors and ubiquitin modification.

To prevent genomic instability disorders, cells have developed a DNA damage response. The response involves various proteins that sense damaged DNA, transduce damage signals, and effect DNA repair. In addition, ubiquitin modifications modulate the signaling pathway depending on cellular context. Among various types of DNA damage, double-stranded breaks are highly toxic to genomic integrity. Homologous recombination (HR) repair is an essential mechanism that fixes DNA damage because of its high level of accuracy. Although factors in the repair pathway are well established, pinpointing the exact mechanisms of repair and devising therapeutic applications requires more studies. Moreover, essential functions of ubiquitin modification in the DNA damage signaling pathway have emerged. In this review, to explore the eukaryotic DNA damage response, we will mention the functions of main factors in the HR repair pathway and ubiquitin modification.

Ring finger protein 126 (RNF126) suppresses ionizing radiation-induced p53-binding protein 1 (53BP1) focus formation.

Cells have evolved sophisticated mechanisms to maintain genomic integrity in response to DNA damage. Ionizing radiation (IR)-induced DNA damage results in the formation of IR-induced foci (iRIF) in the nucleus. The iRIF formation is part of the DNA damage response (DDR), which is an essential signaling cascade that must be strictly regulated because either the loss of or an augmented DDR leads to loss of genome integrity. Accordingly, negative regulation of the DDR is as critical as its activation. In this study, we have identified ring finger protein 126 (RNF126) as a negative regulator of the DDR from a screen of iRIF containing 53BP1. RNF126 overexpression abolishes not only the formation of 53BP1 iRIF but also of RNF168, FK2, RAP80, and BRCA1. However, the iRIF formation of γH2AX, MDC1, and RNF8 is maintained, indicating that RNF126 acts between RNF8 and RNF168 during the DDR. In addition, RNF126 overexpression consistently results in the loss of RNF168-mediated H2A monoubiquitination at lysine 13/15 and inhibition of the non-homologous end joining capability. Taken together, our findings reveal that RNF126 is a novel factor involved in the negative regulation of DDR, which is important for sustaining genomic integrity.

Phosphorylation of Astrin Regulates Its Kinetochore Function.

The error-free segregation of chromosomes, which requires the precisely timed search and capture of chromosomes by spindles during early mitotic and meiotic cell division, is responsible for genomic stability and is achieved by the spindle assembly checkpoint in the metaphase-anaphase transition. Mitotic kinases orchestrate M phase events, such as the reorganization of cell architecture and kinetochore (KT) composition with the exquisite phosphorylation of mitotic regulators, to ensure timely and temporal progression. However, the molecular mechanisms underlying the changes of KT composition for stable spindle attachment during mitosis are poorly understood. Here, we show that the sequential action of the kinase Cdk1 and the phosphatase Cdc14A control spindle attachment to KTs. During prophase, the mitotic spindle protein Spag5/Astrin is transported into centrosomes by Kinastrin and phosphorylated at Ser-135 and Ser-249 by Cdk1, which, in prometaphase, is loaded onto the spindle and targeted to KTs. We also demonstrate that Cdc14A dephosphorylates Astrin, and therefore the overexpression of Cdc14A sequesters Astrin in the centrosome and results in aberrant chromosome alignment. Mechanistically, Plk1 acts as an upstream kinase for Astrin phosphorylation by Cdk1 and targeting phospho-Astrin to KTs, leading to the recruitment of outer KT components, such as Cenp-E, and the stable attachment of spindles to KTs. These comprehensive findings reveal a regulatory circuit for protein targeting to KTs that controls the KT composition change of stable spindle attachment and chromosome integrity.

Overexpression of a novel regulator of p120 catenin, NLBP, promotes lung adenocarcinoma proliferation.

NLBP (novel LZAP-binding protein) was recently shown to function as a tumor suppressor capable of inhibiting the NFκB signaling pathway. NLBP is also known as a negative regulator of cell invasion, and its expression is reduced in several cancer cell lines that have little invasive activity. Although these phenomena suggest that NLBP may be a potential tumor suppressor, its role as a tumor suppressor in human lung cancer is not well established. In contrast to our expectation, NLBP was highly expressed in the early stage of lung adenocarcinoma tissues, and overexpression of NLBP promoted proliferation of H1299 lung adenocarcinoma cells. We also found that p120 catenin (p120ctn) was a novel binding partner of NLBP, and that NLBP binds to the regulatory domain of p120ctn, and p120ctn associates with N-terminal region of NLBP, respectively. This binding leads to p120ctn stability to inhibit proteasomal degradation of p120ctn by inhibiting its ubiqutination. In addition, we also found that overexpression of NLBP and p120ctn in human lung cancer are closely related with adenocarcinoma compared with squamous cell carcinoma. Taken together, our findings reveal that NLBP is highly overexpressed in human lung adenocarcinoma, and that overexpression of NLBP promotes the cell proliferation of lung adenocarcinoma through interacting with p120ctn and suggest that NLBP may function as an oncogene in early stage carcinogenesis of lung adenocarcinoma.

Cdk1 protein-mediated phosphorylation of receptor-associated protein 80 (RAP80) serine 677 modulates DNA damage-induced G2/M checkpoint and cell survival.

Post-translational phosphorylation plays critical roles in the assembly of signaling and repair proteins in the DNA damage response pathway. RAP80, a component of the BRCA1-A complex, is crucial in cell cycle checkpoint activation and DNA damage repair. However, its molecular mechanism is unclear. In this study, we identified Cdk1 as a new RAP80-binding protein and demonstrated that the Cdk1-cyclin B(1) complex phosphorylates RAP80 at Ser-677 using an in vitro kinase assay and a phosphopeptide-specific antibody against phospho-Ser-677 of RAP80. RAP80 Ser-677 phosphorylation occurred in the M phase of the cell cycle when Cdk1 was in an active state. In addition, ionizing radiation (IR) induced RAP80 phosphorylation at Ser-677. Mutation of Ser-677 to alanine sensitized cells to IR and functioned in G(2)/M checkpoint control. These results suggest that post-translational phosphorylation of RAP80 by the Cdk1-cyclin B(1) complex is important for RAP80 functional sensitivity to IR and G(2)/M checkpoint control.

[A case of therapy-related acute monocytic leukemia following low-dose of etoposide treatment for hemophagocytic lymphohistiocytosis].

We report a case of therapy-related acute myeloid leukemia after low-dosed topoisomerase II inhibitor (etoposide) treatment for hemophagocytic lymphohistiocytosis (HLH). A 62-yr-old female patient had previously been treated with a HLH-94 protocol containing a low-dose of etoposide (total dose of 300 mg/m2). Thirty-one months later, the patient was admitted to the hematology department with general weakness and upper respiratory infection symptoms. Peripheral blood smear and bone marrow study revealed acute monocytic leukemia. There was no evidence of myelodysplastic syndrome, and a cytogenetic study showed no chromosomal abnormalities.

Chemesthesis from volatile organic compounds: Psychophysical and neural responses.

In Experiment 1, subjects sought to localize the nostril stimulated, left or right, in tests with nine esters (acetates, propionates, and butyrates) at concentrations meant to trigger chemesthesis (pungency, irritation). The task produced psychometric functions for chemesthetic detection unconfounded by olfactory sensations. The functions indicated a sharp transition from no detection to perfect detection, rather uniform across the esters, which themselves varied in potency by two log units. The correlation between the thresholds for the eight materials that yielded thresholds and predictions from a published linear free energy relationship (LFER) equaled 0.99. In Experiment 2, amplitude of the negative mucosal potential (NMP) was recorded from the septum. The resulting functions also increased with concentration sharply. Against a criterion amplitude of the NMP, thresholds measured in the first experiment (and predictions from the LFER) correlated 0.99. The NMP seems to offer an adequate objective measure of sensory irritation. The LFER, although effective predictively, could stand to have a parameter to anticipate that molecules beyond a certain size fail to trigger irritation. In the present case, a cut-off of chemesthetic potency occurred between butyl butyrate and hexyl butyrate for the group of subjects, with some variation of the boundary among individuals.

Sensory and associated reactions to mineral dusts: sodium borate, calcium oxide, and calcium sulfate.

Occupational exposure limits (OELs) for irritant dusts have had no quantifiable bases. This study (1) charted chemosensory feel, denoted chemesthesis here, to dusts of calcium oxide (1 to 5 mg/m(3)), sodium tetraborate pentahydrate [sodium borate] (5 to 40 mg/m(3)), and calcium sulfate (10 to 40 mg/m(3)); (2) examined correlates of the chemesthetic sensations; and (3) sought to illuminate the basis for potency. Twelve screened men exercised against a light load while they breathed air in a dome fed with controlled levels of dust for 20 min. Measured parameters included nasal resistance, nasal secretion, minute ventilation, heart rate, blood oxygenation, mucociliary transport time, and chemesthetic magnitude, calibrated to pungency of carbon dioxide. Subjects registered time-dependent feel from exposures principally in the nose, secondarily in the throat, and hardly in the eyes. Calcium oxide had the greatest potency, followed by sodium borate, with calcium sulfate a distant third. Of the physiological parameters, amount of secretion showed the best association with chemesthetic potency. That measure, as well as mucociliary transport time and minute ventilation, went into calculation of mass of dust dissolved into mucus. The calculations indicated that the two alkaline dusts increased in equal molar amounts with time. At equal molar concentrations, they had, to a first approximation, equal chemesthetic magnitude. On the basis of mass concentration in air or dissolved into mucus, calcium oxide and sodium borate differed in potency by a factor just above five, equal to the difference in their molecular weights. This relationship could inform the setting of OELs for a critical effect of irritation.

Adsorption and isotope effects by ion exchange with 1-aza-12-crown-4 bonded Merrifield peptide resin.

Magnesium isotope effects were investigated by chemical ion exchange with synthesized 1-aza-12-crown-4 bonded Merrifield peptide resin using elution chromatography. The capacity of azacrown ion exchanger was 0.89 meq/g dry resin. The heavier isotopes of magnesium were enriched in the resin phase, while the lighter isotopes were enriched in the solution phase. The hydration effect is less than the complexation and isotope mass effects. The single stage separation factor was determined according to the method of Glueckauf from the elution curve and isotopic assays. The separation factors of 24Mg(2+)-25Mg(2+), 24Mg(2+)-26Mg(2+), and 25Mg(2+)-26Mg(2+) were 1.012, 1.023, and 1.011, respectively.

Oxaliplatin and UFT combination chemotherapy in patients with metastatic colorectal cancer.

A phase II study was performed to evaluate the clinical efficacy and toxicity of oxaliplatin combined with uracil and tegafur (UFT) in patients with advanced colorectal cancer previously treated with a fluoropyrimidine-based regimen. From January to December 1999, 34 patients were enrolled in this study. Patients received intravenous oxaliplatin 130 mg/m2 on day 1 and daily oral UFT 350 mg/m2 in 3 divided doses for 21 days and repeated every 21 days. Thirty-one of 34 patients were assessable for response and 32 patients for toxicity. Partial response was observed in four patients and stable disease in six patients. The response rate was 12.9% (95% CI, 3.6-29.8%) and median duration of response was 17 weeks. The median overall survival and progression-free survival of all patients were 26 weeks (range, 3-90+ weeks) and 9 weeks (range, 3-56 weeks), respectively. Sensory neuropathy was the most common toxicity, but there was no severe toxicity (>grade II) except for a case of grade III neutropenia. We conclude that oxaliplatin and UFT combination chemotherapy was well tolerated without significant toxicities. The results of this trial will serve as the basis for designing new clinical trials with a different dose or schedule.