NELF and PAF1C complexes are core transcriptional machineries controlling colon cancer stemness.

Mutations in APC, found in 80% of colon caner, enhance ß-catenin stabilization, which is the initial step of colonic tumorigenesis. However, the core transcriptional mechanism underlying the induction of colon cancer stemness by stable ß-catenin remains unclear. Here, we found that inducible inhibition of ß-catenin suppressed elongation of Pol II and RNA polymerase-associated factor 1 complex (PAF1C) around the transcription start site (TSS) of LGR5. Moreover, stable ß-catenin enhanced the formation of active Pol II complex cooperatively with CDC73 and CDK9 by facilitating the recruitment of DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF) complexes to the Pol II complex. Subsequently, stable ß-catenin facilitated the formation of the Pol II-DSIF-PAF1C complex, suggesting that stable ß-catenin induces cancer stemness by stimulating active Pol II complex through NELF and PAF1C. Furthermore, NELF or PAF1C inhibition recapitulated the changes in cancer stemness-related gene expression induced by the inhibition of stable ß-catenin and suppressed colon cancer stemness. Additionally, the chemical inhibition of CDK12 (a downstream transcription CDK of PAF1C) suppressed colon cancer stemness. These results suggest that NELF and PAF1C are the core transcriptional machineries that control expression of colon cancer stemness-inducing genes and may be therapeutic targets for colon cancer.

Blocking LC3 lipidation and ATG12 conjugation reactions by ATG7 mutant protein containing C572S.

Autophagy, a system for the bulk degradation of intracellular components, is essential for homeostasis and the healthy physiology and development of cells and tissues. Its deregulation is associated with human disease. Thus, methods to modulate autophagic activity are critical for analysis of its role in mammalian cells and tissues. Here we report a method to inhibit autophagy using a mutant variant of the protein ATG7, a ubiquitin E1-like enzyme essential for autophagosome formation. During autophagy, ATG7 activates the conjugation of LC3 (ATG8) with phosphatidylethanolamine (PE) and ATG12 with ATG5. Human ATG7 interactions with LC3 or ATG12 require a thioester bond involving the ATG7 cysteine residue at position 572. We generated TetOff cells expressing mutant ATG7 protein carrying a serine substitution of this critical cysteine residue (ATG7C572S). Because ATG7C572S forms stable intermediate complexes with LC3 or ATG12, its expression resulted in a strong blockage of the ATG-conjugation system and suppression of autophagosome formation. Consequently, ATG7C572S mutant protein can be used as an inhibitor of autophagy.

[Transient increase of intraocular pressure with primary open angle glaucoma patients associated with warm and cold seasons after long waiting time in an ophthalmology clinic].

PURPOSE: To retrospectively evaluate the influence of long waiting time on intraocular pressure (IOP) with primary open angle glaucoma (POAG) patients in the waiting room of an ophthalmology clinic. PATIENTS AND METHOD: We reviewed 74 consecutive patients with POAG who visited at Kaimeido Ophthalmic and Dental Clinic within the past 8 years without change of glaucoma medications, 34 men and 40 woman, average age +/- standard deviation) 67.9 +/- 13.4 years. Right eye data of all patients were enrolled and IOP was split into 4 groups, short waiting time (< 30 min ; SH30) and long waiting time (> or = 60 min; LH60) in the hot (> or = 15 degrees C) season. Short waiting time (SC30) and long waiting time (LC60) in the cold (< or = 5 degrees C) season; according to the average outdoor air temperature in Sapporo Japan. All groups were compared each other (paired t test). RESULTS: There was a significant difference between SH30 (14.6 2.4 mmHg) and LH60 (14.9 +/- 2.4 mmHg; p = 0.038), between SC30 (14.6 +/- 2.4 mmHg) and LC60 (15.5 +/- 2.6 mmHg; p = 3.45 x 10(-7)) and between LH60 and LC60 (p = 0.00079). On the other hand, there was no significant difference between SH30 and SC30 (p = 0.89). CONCLUSION: This retrospective study suggests that patients with POAG show a transient IOP elevation after longer waiting time, especially during the cold season.

[Transient elevation of intraocular pressure in primary open-angle glaucoma patients after automated visual field examination in the winter].

PURPOSE: To evaluate retrospectively seasonal fluctuations of transient intraocular pressure (IOP) elevation after automated visual field examination in patients with primary open-angle glaucoma (POAG). PATIENTS AND METHODS: We reviewed 53 consecutive patients with POAG who visited Kaimeido Ophthalmic and Dental Clinic from January 2011 to March 2013, 21 men and 32 women aged 67.7 +/- 11.2 years. The patients were divided into 4 groups, spring, summer, autumn, and winter according to the month of automated visual field examination and both eyes of each patient were enrolled. IOP was measured immediately after automated visual field examination (vf IOP) and compared with the average IOP from the previous 3 months (pre IOP) and with the average IOP from the following 3 months (post IOP) in each season. IOP elevation rate was defined as (vf IOP- pre IOP)/pre IOP x 100% and calculated for each season (paired t test). Additionally, the correlation between mean deviation (MD) and IOP elevation rate was evaluated (single regression analysis). Exclusion criteria were patients who received cataract surgery during this study or had a history of any previous glaucoma surgery. The automated visual field test was performed with a Humphrey field analyzer and the Central 30-2 FASTPAC threshold program. RESULTS: The average vf IOP was 14.5 +/- 2.5 mmHg, higher than pre IOP 13.8 +/- 2.4 mmHg (p < 0.0001) and the post IOP 13.8 +/- 2.2 mmHg (p < 0.0001). IOP elevation rate in each season was 4.1 11.6% in spring (n = 22, p = 0.18), 0.1 +/- 9.9% in summer (n = 16, p = 1.0), 5.0 +/- 13.8% in autumn (n = 30. p = 0.11), 10.6 +/- 8.8% in winter (n = 38, p < 0.0001). The MD was not correlated with IOP elevation rate (p = 0.17). CONCLUSION: Patients with POAG show a transient IOP elevation after automated visual field examination, especially in the winter but not in the summer.

Reduction of intraocular pressure using a modified 360-degree suture trabeculotomy technique in primary and secondary open-angle glaucoma: a pilot study.

PURPOSE: To investigate the effects of a modified 360-degree suture trabeculotomy technique for primary and secondary open-angle glaucoma (POAG and SOAG). METHODS: We modified the procedure for 360-degree trabeculotomy by using a 5-0 nylon suture, making a scleral flap to allow clear identification of Schlemm canal, and creating a corneal side port incision opposite to the scleral flap to retrieve the suture used to cannulate and cleave the canal. The modified 360-degree suture trabeculotomy (not combined with cataract surgery) was performed on 25 eyes with POAG and 18 eyes with SOAG, and the results were compared retrospectively with those of standard trabeculotomy with metal trabeculotomes (16 eyes with POAG and 19 eyes with SOAG). When the intraocular pressure (IOP) was reduced by 30% from the preoperative IOP and was also below 18 mm Hg at 3, 6, 9, 12, and 18 months after surgery and the patient was taking a similar number or fewer medications, the surgery was considered a "success." RESULTS: Using this modified technique, Schlemm canal was appropriately incised without resistance. At 12 months after the modified 360-degree suture trabeculotomy and trabeculotomy with metal trabeculotomes, the mean postoperative IOP values were 13.1 and 15.2 mm Hg, respectively, and the mean numbers of antiglaucoma medications were 0.5 and 1.4, respectively. The success rates of POAG at 12 months for the modified 360-degree suture trabeculotomy and trabeculotomy with metal trabeculotomes were 84% and 31%, respectively, and those of SOAG were 89% and 50%, respectively. The complications included a transient elevation of the IOP above 30 mm Hg in 22 eyes (47%) treated with the 360-degree suture trabeculotomy and 17 eyes (49%) treated with trabeculotomy with metal trabeculotomes. There was no significant difference between preoperative visual acuity and postoperative visual acuity in either procedure. CONCLUSIONS: This modified 360-degree suture trabeculotomy is a feasible surgical option for POAG and SOAG.