The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery.

Checkpoint activation after DNA damage causes a transient cell cycle arrest by suppressing cyclin-dependent kinases (CDKs). However, it remains largely elusive how cell cycle recovery is initiated after DNA damage. In this study, we discovered the upregulated protein level of MASTL kinase hours after DNA damage. MASTL promotes cell cycle progression by preventing PP2A/B55-catalyzed dephosphorylation of CDK substrates. DNA damage-induced MASTL upregulation was caused by decreased protein degradation, and was unique among mitotic kinases. We identified E6AP as the E3 ubiquitin ligase that mediated MASTL degradation. MASTL degradation was inhibited upon DNA damage as a result of the dissociation of E6AP from MASTL. E6AP depletion reduced DNA damage signaling, and promoted cell cycle recovery from the DNA damage checkpoint, in a MASTL-dependent manner. Furthermore, we found that E6AP was phosphorylated at Ser-218 by ATM after DNA damage and that this phosphorylation was required for its dissociation from MASTL, the stabilization of MASTL, and the timely recovery of cell cycle progression. Together, our data revealed that ATM/ATR-dependent signaling, while activating the DNA damage checkpoint, also initiates cell cycle recovery from the arrest. Consequently, this results in a timer-like mechanism that ensures the transient nature of the DNA damage checkpoint.

The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery.

Checkpoint activation after DNA damage causes a transient cell cycle arrest by suppressing CDKs. However, it remains largely elusive how cell cycle recovery is initiated after DNA damage. In this study, we discovered the upregulated protein level of MASTL kinase hours after DNA damage. MASTL promotes cell cycle progression by preventing PP2A/B55-catalyzed dephosphorylation of CDK substrates. DNA damage-induced MASTL upregulation was caused by decreased protein degradation, and was unique among mitotic kinases. We identified E6AP as the E3 ubiquitin ligase that mediated MASTL degradation. MASTL degradation was inhibited upon DNA damage as a result of the dissociation of E6AP from MASTL. E6AP depletion reduced DNA damage signaling, and promoted cell cycle recovery from the DNA damage checkpoint, in a MASTL-dependent manner. Furthermore, we found that E6AP was phosphorylated at Ser-218 by ATM after DNA damage and that this phosphorylation was required for its dissociation from MASTL, the stabilization of MASTL, and the timely recovery of cell cycle progression. Together, our data revealed that ATM/ATR-dependent signaling, while activating the DNA damage checkpoint, also initiates cell cycle recovery from the arrest. Consequently, this results in a timer-like mechanism that ensures the transient nature of the DNA damage checkpoint.

The MASTL-ENSA-PP2A/B55 axis modulates cisplatin resistance in oral squamous cell carcinoma.

Platinum-based chemotherapy is the standard first-line treatment for oral squamous cell carcinoma (OSCC) that is inoperable, recurrent, or metastatic. Platinum sensitivity is a major determinant of patient survival in advanced OSCC. Here, we investigated the involvement of MASTL, a cell cycle kinase that mediates ENSA/ARPP19 phosphorylation and PP2A/B55 inhibition, in OSCC therapy. Interestingly, upregulation of MASTL and ENSA/ARPP19, and downregulation of PP2A/B55, were common in OSCC. MASTL expression was in association with poor patient survival. In established OSCC cell lines, upregulation of MASTL and ENSA, and downregulation of B55 genes, correlated with cisplatin resistance. We further confirmed that stable expression of MASTL in OSCC cells promoted cell survival and proliferation under cisplatin treatment, in an ENSA-dependent manner. Conversely, deletion of MASTL or ENSA, or overexpression of B55α, sensitized cisplatin response, consistent with increased DNA damage accumulation, signaling, and caspase activation. Moreover, GKI-1, the first-in-class small molecule inhibitor of MASTL kinase, phenocopied MASTL depletion in enhancing the outcome of cisplatin treatment in OSCC cells, at a dose substantially lower than that needed to disrupt mitotic entry. Finally, GKI-1 exhibited promising efficacy in a mouse tumor xenograft model, in conjunction with cisplatin therapy.

The phosphatase CTDSPL2 is phosphorylated in mitosis and a target for restraining tumor growth and motility in pancreatic cancer.

Carboxy-terminal domain (CTD) small phosphatase like 2 (CTDSPL2), also known as SCP4 or HSPC129, is a new member of the small CTD phosphatase (SCP) family and its role in cancers remains unclear. Here, we used a Phos-tag technique to screen a series of phosphatases and identified CTDSPL2 as a mitotic regulator. We demonstrated that CTDSPL2 was phosphorylated at T86, S104, and S134 by cyclin-dependent kinase 1 (CDK1) in mitosis. Depletion of CTDSPL2 led to mitotic defects and prolonged mitosis. Resultantly, CTDSPL2 deletion restrained proliferation, migration, and invasion in pancreatic cancer cells. We further confirmed the dominant negative effects of a phosphorylation-deficient mutant form of CTDSPL2, implying the biological significance of CTDSPL2 mitotic phosphorylation. Moreover, RT2 cell cycle array analysis revealed p21 and p27 as downstream regulators of CTDSPL2, and inhibition of p21 and/or p27 partially rescued the phenotype in CTDSPL2-deficient cell lines. Importantly, both CTDSPL2 depletion and phosphorylation-deficient mutant CTDSPL2 hindered tumor growth in xenograft models. Together, our findings for the first time highlight the novel role of CTDSPL2 in regulating cell mitosis, proliferation and motility in pancreatic cancer and point out the implications of CTDSPL2 in regulating two critical cell cycle participants (p21 and p27), providing an alternative molecular target for pancreatic cancer treatment.

The Sm core components of small nuclear ribonucleoproteins promote homologous recombination repair.

DNA Double strand breaks (DSBs) are highly hazardous to the cell, and are repaired predominantly via non-homologous end joining (NHEJ) and homologous recombination (HR). Using DSB-mimicking DNA templates, our proteomic studies identified a group of Sm core proteins of small nuclear ribonucleoproteins (snRNPs) as potential DSB-associated proteins. We further confirmed that these Sm proteins were recruited to laser-induced DNA damage sites, and co-localized with established DNA damage repair factors. Depletion of Sm-D3 or Sm-B induced accumulation of γ-H2AX, and impaired the repair efficiency of HR, but not NHEJ. Furthermore, disruption of Sm-D3 reduced the protein level of HR factors, especially RAD51 and CHK1, but caused no change in the expression of repair factors involved in NHEJ. Mechanistically, Sm-D3 proteins bound RAD51, suppressed the ubiquitination of RAD51, and mediated the stabilization of RAD51; Sm-D3 depletion particularly impacted the level of RAD51 and CHK1 on damaged chromatin. As such, our studies characterized a role of Sm proteins in HR repair, via a new mechanism that is distinct from their conventional functions in RNA processing and gene regulation, but consistent with their direct recruitment to DNA damage sites and association with repair factors.

Discovery of Halogenated Benzothiadiazine Derivatives with Anticancer Activity*.

Mitochondrial respiratory complex II (CII), also known as succinate dehydrogenase, plays a critical role in mitochondrial metabolism. Known but low potency CII inhibitors are selectively cytotoxic to cancer cells including the benzothiadiazine-based anti-hypoglycemic diazoxide. Herein, we study the structure-activity relationship of benzothiadiazine derivatives for CII inhibition and their effect on cancer cells for the first time. A 15-fold increase in CII inhibition was achieved over diazoxide, albeit with micromolar IC50 values. Cytotoxicity evaluation of the novel derivatives resulted in the identification of compounds with much greater antineoplastic effect than diazoxide, the most potent of which possesses an IC50 of 2.93±0.07 µM in a cellular model of triple-negative breast cancer, with high selectivity over nonmalignant cells and more than double the potency of the clinical agent 5-fluorouracil. No correlation between cytotoxicity and CII inhibition was found, thus indicating an as-yet-undefined mechanism of action of this scaffold. The derivatives described herein represent valuable hit compounds for therapeutic discovery in triple-negative breast cancer.

PARP1 Upregulation in Recurrent Oral Cancer and Treatment Resistance.

First-line treatments for oral cancer typically include surgery, radiation, and in some cases, chemotherapy. Radiation and oral cancer chemotherapeutics confer cytotoxicity largely by inducing DNA damage, underscoring the importance of the cellular DNA damage repair and response pathways in cancer therapy. However, tumor recurrence and acquired resistance, following the initial response to treatment, remains as a major clinical challenge. By analyzing oral tumor cells derived from the primary and recurrent tumors of the same patient, our study revealed upregulated PARP1 expression in the recurrent tumor cells. Cisplatin and 5-fluorouracil treatment further augmented PARP1 expression in the recurrent, but not the primary, tumor cells. Post-treatment upregulation of PARP1 was dependent on the catalytic activities of PARP and CDK7. Consistent with the established function of PARP1 in DNA repair, we showed that overexpression of PARP1 rendered the primary tumor cells highly resistant to DNA damage treatment. Conversely, PARP inhibition partially reversed the treatment resistance in the recurrent tumor cells; combinatorial treatment using a PARP inhibitor and cisplatin/5-fluorouracil significantly sensitized the tumor response in vivo. Taken together, we reported here PARP1 upregulation as a clinically relevant mechanism involved in oral cancer recurrence, and suggested the clinical benefit of PARP inhibitors, currently approved for the treatment of several other types of cancer, in oral cancer.

Kinesin Kif2C in regulation of DNA double strand break dynamics and repair.

DNA double strand breaks (DSBs) have detrimental effects on cell survival and genomic stability, and are related to cancer and other human diseases. In this study, we identified microtubule-depolymerizing kinesin Kif2C as a protein associated with DSB-mimicking DNA templates and known DSB repair proteins in Xenopus egg extracts and mammalian cells. The recruitment of Kif2C to DNA damage sites was dependent on both PARP and ATM activities. Kif2C knockdown or knockout led to accumulation of endogenous DNA damage, DNA damage hypersensitivity, and reduced DSB repair via both NHEJ and HR. Interestingly, Kif2C depletion, or inhibition of its microtubule depolymerase activity, reduced the mobility of DSBs, impaired the formation of DNA damage foci, and decreased the occurrence of foci fusion and resolution. Taken together, our study established Kif2C as a new player of the DNA damage response, and presented a new mechanism that governs DSB dynamics and repair.

DNA can be damaged in many ways, and a double strand break is one of the most dangerous. This occurs when both strands of the double helix snap at the same time, leaving two broken ends. When cells detect this kind of damage, they race to get it fixed as quickly as possible. Fixing these double strand breaks is thought to involve the broken ends being moved to 'repair centers' in the nucleus of the cell, but it was unclear how the broken ends were moved. One possibility was that the cells transport the broken ends along protein filaments called microtubules. Cells can assemble these track-like filaments on-demand to carry cargo attached to molecular motors called kinesins. However, this type of transport happens outside of the cell's nucleus, and while there are different kinesin proteins localized inside the nucleus, their roles are largely unknown. In an effort to understand how broken DNA ends are repaired, Zhu, Paydar et al. conducted experiments that simulated double strand breaks and examined the proteins that responded. The first set of experiments involved mixing cut pieces of DNA with extracts taken from frog eggs or human cells. Zhu, Paydar et al. found that one kinesin called Kif2C stuck to the DNA fragments, and attached to many proteins known to play a role in DNA damage repair. Kif2C had previously been shown to help separate the chromosomes during cell division. To find out more about its potential role in DNA repair, Zhu, Paydar et al. then used a laser to create breaks in the DNA of living human cells and tracked Kif2C movement. The kinesin arrived within 60 seconds of the DNA damage and appeared to transport the cut DNA ends to 'repair centers'. Getting rid of Kif2C, or blocking its activity, had dire effects on the cells' abilities to mobilize and repair breaks to its DNA. Without the molecular motor, fewer double strand breaks were repaired, and so DNA damage started to build up. Defects in double strand break repair happen in many human diseases, including cancer. Many cancer treatments damage the DNA of cancer cells, sometimes in combination with drugs that stop cells from building and using their microtubule transport systems. Understanding the new role of Kif2C in DNA damage repair could therefore help optimize these treatment combinations.

[Minimally invasive external fixation of humeral supracondylar angular rotation center for the treatment of cubitus varus deformity in adolescents].

OBJECTIVE: To investigate clinical effect of minimally invasive osteotomy and external fixation with the center of roration of angulation (CORA) in treating cubitus varus in adolescents. METHODS: From August 2013 to August 2017, 15 patients with cubitus varus caused by supracondylar fracture of humerus were treated with minimally invasive osteotomy and external fixation with the CORA. Among them, including 9 males and 6 females; 11 patients on the left side and 4 patients on the right side; aged from 13 to 16 years old with an average of 14.5 years old. The time from injury to operation was for 6 to 10 years with an average of 7.5 years. Five patients had a history of recurrence after cubitus varus surgery. Correction time. fracture healing time, carrying angle were observed, Laupattarakasem standard was used to evaluate clinical effect. RESULTS: All patients were followed up from 12 to 30 months with an average of 24 months; correction time ranged from 3 to 5 weeks with an average of 4 weeks; fracture healing time ranged from 4 to 6 months with an average of 5 months; carrying angle before operation ranged from -12° to -23°, and improved 9° to 14° after operation. According to Laupattarakasem evaluation criteria, 11 patients got an excellent result, 3 good and 1 fair. CONCLUSIONS: Minimally invasive osteotomy and external fixation with CORA in treating cubitus varus deformity in adolescents has advantages of less trauma, less blood loss, earlier exercise, speed and angle of correction could controlled without hospitalized for fixation.

[Modified Ilizarov hip reconstruction in treatment of adolescent hip instability].

OBJECTIVE: To evaluate the effectiveness of modified Ilizarov hip reconstruction in the treatment of hip instability. METHODS: The clinical data of 13 young patients with hip diseases treated with modified Ilizarov hip reconstruction between January 2010 and March 2018 were retrospectively analyzed. There were 2 males and 11 females, aged from 14 to 34 years, with an average age of 24.2 years. There were 1 case of hip dysplasia and dislocation due to spinal bifida, 3 cases of hip dysplasia after pyogenic arthritis of the hip, 2 cases of developmental dysplasiaof the hip (DDH) accompanying femoral head necrosis who rejected hip replacement, 6 cases of young DDH refused to undergo hip replacement, and 1 case of bilateral hip dysplasia with dislocation due to sputum cerebral palsy. The disease duration was 2-20 years, with an average of 8.5 years. Preoperative Trendelenburg sign was positive in 12 cases and negative in 1 case. The preoperative Harris score of hip joint was 53.5±8.9 and the unequal length of lower limbs was (46.08±15.73) mm. Postoperative Harris hip score and patients' satisfaction with effectiveness evaluated according to their self scoring were used to assess the effectiveness. RESULTS: All 13 patients were followed up 1-5 years, with an average of 2.6 years. Five patients developed postoperative needle infection, which improved after dressing change; 7 patients had limited knee joint activity and improved after knee joint function training. The Trendelenburg sign was negative at 1 year after operation, and the patient's hip pain symptoms were relieved or disappeared. The Harris hip score of patients at 1 year after operation was 84.5±6.1, which was significantly improved when compared with preoperative one ( t=－10.538, P=0.000). According to Harris hip score, the effectiveness results were excellent in 4 cases, good in 5 cases, and fair in 4 cases, with an excellent and good rate of 69.2%. The unequal length of lower limbs was (15.38±7.27) mm, which was significantly better than that before operation ( t=11.826, P=0.000). At last follow-up, the patients' satisfaction score was 80%-95%, with an average of 88%. CONCLUSION: Modified Ilizarov hip reconstruction can be used to treat young patients with hip disease who are unsuitable or refuse to undergo artificial hip replacement. Its effectiveness is reliable, and it has unique advantages in limb limp improvement and limb shortening correction.

Phosphatase 1 Nuclear Targeting Subunit Mediates Recruitment and Function of Poly (ADP-Ribose) Polymerase 1 in DNA Repair.

PARP, particularly PARP1, plays an essential role in the detection and repair of DNA single-strand breaks and double-strand breaks. PARP1 accumulates at DNA damage sites within seconds after DNA damage to catalyze the massive induction of substrate protein poly ADP-ribosylation (PARylation). However, the molecular mechanisms underlying the recruitment and activation of PARP1 in DNA repair are not fully understood. Here we show that phosphatase 1 nuclear targeting subunit 1 (PNUTS) is a robust binding partner of PARP1. Inhibition of PNUTS led to strong accumulation of endogenous DNA damage and sensitized the cellular response to a wide range of DNA-damaging agents, implicating PNUTS as an essential and multifaceted regulator of DNA repair. Recruitment of PNUTS to laser-induced DNA damage was similar to that of PARP1, and depletion or inhibition of PARP1 abrogated recruitment of PNUTS to sites of DNA damage. Conversely, PNUTS was required for efficient induction of substrate PARylation after DNA damage. PNUTS bound the BRCA1 C-terminal (BRCT) domain of PARP1 and was required for the recruitment of PARP1 to sites of DNA damage. Finally, depletion of PNUTS rendered cancer cells hypersensitive to PARP inhibition. Taken together, our study characterizes PNUTS as an essential partner of PARP1 in DNA repair and a potential drug target in cancer therapy. SIGNIFICANCE: These findings reveal PNUTS as an essential functional partner of PARP1 in DNA repair and suggest its inhibition as a potential therapeutic strategy in conjunction with DNA-damaging agents or PARP inhibitors.See related commentary by Murai and Pommier, p. 2460.

Phosphatase 1 Nuclear Targeting Subunit (PNUTS) Regulates Aurora Kinases and Mitotic Progression.

Mitotic progression is regulated largely by reversible phosphorylation events that are mediated by mitotic kinases and phosphatases. Protein phosphatase 1 (PP1) has been shown to play a crucial role in regulation of mitotic entry, progression, and exit. We previously observed, in Xenopus egg extracts, that phosphatase 1 nuclear targeting subunit (PPP1R10/PNUTS) acts as a mitotic regulator by negatively modulating PP1. This study investigates the role of PNUTS in mitotic progression in mammalian cells, and demonstrates that PNUTS expression is elevated in mitosis and depletion partially blocks mitotic entry. Cells that enter mitosis after PNUTS knockdown exhibit frequent chromosome mis-segregation. Aurora A/B kinase complexes and several kinetochore components are identified as PNUTS-associated proteins. PNUTS depletion suppresses the activation of Aurora A/B kinases, and disrupts the spatiotemporal regulation of the chromosomal passenger complex (CPC). PNUTS dynamically localizes to kinetochores, and is required for the activation of the spindle assembly checkpoint. Finally, PNUTS depletion sensitizes the tumor cell response to Aurora inhibition, suggesting that PNUTS is a potential drug target in combination anticancer therapy. IMPLICATIONS: Delineation of how PNUTS governs the mitotic activation and function of Aurora kinases will improve the understanding of the complex phospho-regulation in mitotic progression, and suggest new options to enhance the therapeutic efficacy of Aurora inhibitors.

[Effectiveness of minimally invasive osteotomy Ilizarov technique combined with intramedullary nail for femoral lengthening].

Objective: To explore the effectiveness of minimally invasive osteotomy Ilizarov technique combined with intramedullary nail for femoral lengthening. Methods: Seventy-one patients with femoral shortening deformity who met the selection criteria between January 2013 and June 2016 were randomly divided into trial group (36 cases were treated with minimally invasive osteotomy Ilizarov technique combined with intramedullary nail for femoral lengthening) and control group (35 cases were treated with simple Ilizarov technique for femoral lengthening). There was no significant difference in age, gender, causes of femoral shortening, length of femoral shortening, rate of femoral deformity between the two groups ( P>0.05). The operation duration, intraoperative blood loss, lengthening rate, external fixation duration, frequency of pin tract infection, osteotomy healing time, and range of motion (ROM) of knee at 1 year after operation were recorded and compared between the two groups. Results: The patients of two groups were followed up 12-60 months (mean, 31 months). Pin tract infection occured in 8 cases (10 pins), including 1 case (1 pin) in the trial group and 7 cases (9 pins) in the control group. There was significant difference in the incidence of pin tract infection between the two groups ( χ2=5.265, P=0.022). All patients were cured by replacing the fixation pins, changing dressing actively, application of antibiotics, and adequate postoperative care. The operation duration, intraoperative blood loss, external fixation duration, osteotomy healing time, and ROM of knee at 1 year after operation of the trial group were superior to those of the control group, showing significant differences ( P<0.05). There was no significant difference in the lengthening rate between the two groups ( t=-1.581, P=0.153). Conclusion: The minimally invasive osteotomy Ilizarov technique combined with intramedullary nail in femoral lengthening increases the operation time, but the external fixation duration and incidence of pin tract infection are significantly reduced and the function of knee is significantly improved.

Protein interactomes of protein phosphatase 2A B55 regulatory subunits reveal B55-mediated regulation of replication protein A under replication stress.

The specific function of PP2A, a major serine/threonine phosphatase, is mediated by regulatory targeting subunits, such as members of the B55 family. Although implicated in cell division and other pathways, the specific substrates and functions of B55 targeting subunits are largely undefined. In this study we identified over 100 binding proteins of B55α and B55ß in Xenopus egg extracts that are involved in metabolism, mitochondria function, molecular trafficking, cell division, cytoskeleton, DNA replication, DNA repair, and cell signaling. Among the B55α and B55ß-associated proteins were numerous mitotic regulators, including many substrates of CDK1. Consistently, upregulation of B55α accelerated M-phase exit and inhibited M-phase entry. Moreover, specific substrates of CDK2, including factors of DNA replication and chromatin remodeling were identified within the interactomes of B55α and B55ß, suggesting a role for these phosphatase subunits in DNA replication. In particular, we confirmed in human cells that B55α binds RPA and mediates the dephosphorylation of RPA2. The B55-RPA association is disrupted after replication stress, consistent with the induction of RPA2 phosphorylation. Thus, we report here a new mechanism that accounts for both how RPA phosphorylation is modulated by PP2A and how the phosphorylation of RPA2 is abruptly induced after replication stress.

Protein phosphatase 1 and phosphatase 1 nuclear targeting subunit-dependent regulation of DNA-dependent protein kinase and non-homologous end joining.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays a key role in mediating non-homologous end joining (NHEJ), a major repair pathway for DNA double-strand breaks (DSBs). The activation, function and dynamics of DNA-PKcs is regulated largely by its reversible phosphorylation at numerous residues, many of which are targeted by DNA-PKcs itself. Interestingly, these DNA-PKcs phosphorylation sites function in a distinct, and sometimes opposing manner, suggesting that they are differentially regulated via complex actions of both kinases and phosphatases. In this study we identified several phosphatase subunits as potential DSB-associated proteins. In particular, protein phosphatase 1 (PP1) is recruited to a DSB-mimicking substrate in Xenopus egg extracts and sites of laser microirradiation in human cells. Depletion of PP1 impairs NHEJ in both Xenopus egg extracts and human cells. PP1 binds multiple motifs of DNA-PKcs, regulates DNA-PKcs phosphorylation, and is required for DNA-PKcs activation after DNA damage. Interestingly, phosphatase 1 nuclear targeting subunit (PNUTS), an inhibitory regulator of PP1, is also recruited to DNA damage sites to promote NHEJ. PNUTS associates with the DNA-PK complex and is required for DNA-PKcs phosphorylation at Ser-2056 and Thr-2609. Thus, PNUTS and PP1 together fine-tune the dynamic phosphorylation of DNA-PKcs after DNA damage to mediate NHEJ.

Cell cycle-dependent regulation of Greatwall kinase by protein phosphatase 1 and regulatory subunit 3B.

Greatwall (Gwl) kinase plays an essential role in the regulation of mitotic entry and progression. Mitotic activation of Gwl requires both cyclin-dependent kinase 1 (CDK1)-dependent phosphorylation and its autophosphorylation at an evolutionarily conserved serine residue near the carboxyl terminus (Ser-883 in Xenopus). In this study we show that Gwl associates with protein phosphatase 1 (PP1), particularly PP1γ, which mediates the dephosphorylation of Gwl Ser-883. Consistent with the mitotic activation of Gwl, its association with PP1 is disrupted in mitotic cells and egg extracts. During mitotic exit, PP1-dependent dephosphorylation of Gwl Ser-883 occurs prior to dephosphorylation of other mitotic substrates; replacing endogenous Gwl with a phosphomimetic S883E mutant blocks mitotic exit. Moreover, we identified PP1 regulatory subunit 3B (PPP1R3B) as a targeting subunit that can direct PP1 activity toward Gwl. PPP1R3B bridges PP1 and Gwl association and promotes Gwl Ser-883 dephosphorylation. Consistent with the cell cycle-dependent association of Gwl and PP1, Gwl and PPP1R3B dissociate in M phase. Interestingly, up-regulation of PPP1R3B facilitates mitotic exit and blocks mitotic entry. Thus, our study suggests PPP1R3B as a new cell cycle regulator that functions by governing Gwl dephosphorylation.

Non-homologous end joining repair in Xenopus egg extract.

Non-homologous end joining (NHEJ) is a major DNA double-strand break (DSB) repair mechanism. We characterized here a series of plasmid-based DSB templates that were repaired in Xenopus egg extracts via the canonical, Ku-dependent NHEJ pathway. We showed that the template with compatible ends was efficiently repaired without end processing, in a manner that required the kinase activity of DNA-PKcs but not ATM. Moreover, non-compatible ends with blunt/3'-overhang, blunt/5'-overhang, and 3'-overhang/5'-overhang were predominantly repaired with fill-in and ligation without the removal of end nucleotides. In contrast, 3'-overhang/3'-overhang and 5'-overhang/5'-overhang templates were processed by resection of 3-5 bases and fill-in of 1-4 bases prior to end ligation. Therefore, the NHEJ machinery exhibited a strong preference for precise repair; the presence of neither non-compatible ends nor protruding single strand DNA sufficiently warranted the action of nucleases. ATM was required for the efficient repair of all non-compatible ends including those repaired without end processing by nucleases, suggesting its role beyond phosphorylation and regulation of Artemis. Finally, dephosphorylation of the 5'-overhang/3'-overhang template reduced the efficiency of DNA repair without increasing the risk of end resection, indicating that end protection via prompt end ligation is not the sole mechanism that suppresses the action of nucleases.

Cell fate determination in cisplatin resistance and chemosensitization.

Understanding the determination of cell fate choices after cancer treatment will shed new light on cancer resistance. In this study, we quantitatively analyzed the individual cell fate choice in resistant UM-SCC-38 head and neck cancer cells exposed to cisplatin. Our study revealed a highly heterogeneous pattern of cell fate choices in UM-SCC-38 cells, in comparison to that of the control, non-tumorigenic keratinocyte HaCaT cells. In both UM-SCC-38 and HaCaT cell lines, the majority of cell death occurred during the immediate interphase without mitotic entry, whereas significant portions of UM-SCC-38 cells survived the treatment via either checkpoint arrest or checkpoint slippage. Interestingly, checkpoint slippage occurred predominantly in cells treated in late S and G2 phases, and cells in M-phase were hypersensitive to cisplatin. Moreover, although the cisplatin-resistant progression of mitosis exhibited no delay in general, prolonged mitosis was correlated with the induction of cell death in mitosis. The finding thus suggested a combinatorial treatment using cisplatin and an agent that blocks mitotic exit. Consistently, we showed a strong synergy between cisplatin and the proteasome inhibitor Mg132. Finally, targeting the DNA damage checkpoint using inhibitors of ATR, but not ATM, effectively sensitized UM-SCC-38 to cisplatin treatment. Surprisingly, checkpoint targeting eliminated both checkpoint arrest and checkpoint slippage, and augmented the induction of cell death in interphase without mitotic entry. Taken together, our study, by profiling cell fate determination after cisplatin treatment, reveals new insights into chemoresistance and suggests combinatorial strategies that potentially overcome cancer resistance.

[Repairing tibial post-traumatic osteomyelitis with bone and skin defect by Ilizarov technique at stage I].

OBJECTIVE: To explore clinical effects of Ilizarov technique at stage I for repairing tibial post-traumatic osteomyelitis with bone and skin defect. METHODS: From June 2010 to December 2013,44 patients with tibial post-traumatic osteomyelitis with bone and skin defect were treated with Ilizarov technique at stage I . Among them, there were 35 males and 9 females aged from 18 to 70 years old with an average of 42.5 years old. Bone defect ranged from 4 to 16 cm, skin defect ranged from 3 cm x 4 cm to 5 cm x 16 cm. The operation was performed debridement thoroughly, removed inflammatory bone section, osteotomy invasively, install circular external fixator by Ilizarow technique; screw nut were rotated at 1 week after operation, and prolonged 0.5 to 1.0 mm everyday. Wound surface, new born callus and bone healing were observed to evaluate clinical effects. RESULTS: All patients were followed up from 11 to 36 months with an average of 18.5 months. Bone defect after osteotomy was from 6 to 22 cm with an average of 11.5 cm; the time of wound healing time ranged from 21 to 79 d with an average of 38 d; bone defect healing time was from 8 to 15 months with an average of 12.5 months. All patients were cured, no recurrent infection, refracture and shorten of calf deformity were occurred. CONCLUSION: Repairing tibial post-traumatic osteomyelitis with bone and skin defect by llizarov technique at stage I has advantages of less trauma, low inflammatory recurrence rate, could avoid multiple complex operation, and receive definite curative effect.

[Ilizarov TECHNOLOGY COMBINED WITH TARSAL V-SHAPE OSTEOTOMY FOR TREATMENT OF TRAUMATIC CLUBFOOT].

OBJECTIVE: To discuss the effectiveness of Ilizarov technology combined with tarsal V-shape osteotomy for the treatment of traumatic clubfoot. METHODS: Between August 2011 and August 2014, 14 patients with traumatic clubfoot were treated. There were 10 males and 4 females, aged 13 to 61 years (mean, 31 years). of 14 cases, 11 had open fractures of the tibia and ankle and 3 had closed fracture of the ankle joint. The interval from trauma to operation was 7-78 months (mean, 36 months). The plantar flexion of the ankle was 44-89° (mean, 57°). After invasive foot soft tissue release and tarsal V-shape osteotomy, the Ilizaroy external fixator with elastic stretching rod was used. At 5-12 weeks after operation, the neutra pos ofthe ankle joint was restored. Then the neutral position of the ankle joint was maintained for 8 to 12 weeks. After removal of external fixator, protective walking brace was used for 8 to 12 weeks. RESULTS: Infection occurred in 9 cases, and was cured after symptomatic treatment. The patients were followed up 10-36 months (mean, 15 months). After treatment, 14 patients had normal appearance of the ankle joint, and X-ray films showed normal structure of the ankle. The ankle dorsal extension was 10° in 9 patients, who had normal walking function; it was 5° in 4 patients, who could walk; in 1 case of neutral position, the foot had no function of up and down stairs. One case had pain during correction because of poor tolerance, and delay traction was given, the function was recovered to normal after active rehabilitation training. According to the International Clubfoot Study Group (ICFSG) score standard, the results were excellent in 9 cases, good in 4 cases, and fair in 1 case; the excellent and good rate was 92.9% at last follow-up. CONCLUSION: Ilizarov external fixation combined with V-shape osteotomy is effective for the treatment of traumatic clubfoot, with the advantages of less trauma, reliable fixation, satisfactory correction of the deformity, and good function recovery of the ankle.