GSK3ß controls epithelial-mesenchymal transition and tumor metastasis by CHIP-mediated degradation of Slug.

This corrects the article DOI: 10.1038/onc.2013.279.

Slug is temporally regulated by cyclin E in cell cycle and controls genome stability.

The transcriptional repressor Slug is best known to control epithelial-mesenchymal transition (EMT) and promote cancer invasion/metastasis. In this study, we demonstrate that Slug is temporally regulated during cell cycle progression. At G1/S transition, cyclin E-cyclin-dependent kinase 2 mediates the phosphorylation of Slug at Ser-54 and Ser-104, resulting in its ubiquitylation and degradation. Non-phosphorylatable Slug is markedly stabilized at G1/S transition compared with wild-type Slug and greatly leads to downregulation of DNA synthesis and checkpoint-related proteins, including TOP1, DNA Ligase IV and Rad17, reduces cell proliferation, delays S-phase progression and contributes to genome instability. Our results indicate that Slug has multifaceted roles in cancer progression by controlling both EMT and genome stability.

GSK3ß controls epithelial-mesenchymal transition and tumor metastasis by CHIP-mediated degradation of Slug.

Glycogen synthase kinase 3 beta (GSK3ß) is highly inactivated in epithelial cancers and is known to inhibit tumor migration and invasion. The zinc-finger-containing transcriptional repressor, Slug, represses E-cadherin transcription and enhances epithelial-mesenchymal transition (EMT). In this study, we find that the GSK3ß-pSer9 level is associated with the expression of Slug in non-small cell lung cancer. GSK3ß-mediated phosphorylation of Slug facilitates Slug protein turnover. Proteomic analysis reveals that the carboxyl terminus of Hsc70-interacting protein (CHIP) interacts with wild-type Slug (wtSlug). Knockdown of CHIP stabilizes the wtSlug protein and reduces Slug ubiquitylation and degradation. In contrast, nonphosphorylatable Slug-4SA is not degraded by CHIP. The accumulation of nondegradable Slug may further lead to the repression of E-cadherin expression and promote cancer cell migration, invasion and metastasis. Our findings provide evidence of a de novo GSK3ß-CHIP-Slug pathway that may be involved in the progression of metastasis in lung cancer.

ATXN8 -62 G/A promoter polymorphism and risk of Taiwanese Parkinson's disease.

BACKGROUND AND PURPOSE: We recently reported a novel -62 G/A polymorphism within ataxin 8 (ATXN8) gene promoter region, with -62 G displaying significantly higher luciferase activity compared with -62 A. Phenotypic variability in spinocerebellar ataxia type 8 (SCA8) has been suggested, and large SCA8 repeats were found in patients with Parkinson's disease (PD). We aimed to investigate the association of ATXN8 -62 G/A polymorphism with the risk of Taiwanese PD, and identify the trans-acting factor modulating the ATXN8 promoter activity. METHODS: A case-control study in a cohort of 569 PD cases and 547 ethnically matched controls was conducted by polymerase chain reaction (PCR) and restriction enzyme analysis. The trans-acting factor binding to the ATXN8 promoter was examined by chromatin immunoprecipitation (ChIP)-PCR assay, cDNA co-transfection and luciferase reporter assay. RESULTS: When genotype distribution was calculated by comparing the rare AA genotype with the GG + GA genotypes (recessive model), a significant difference was found (P = 0.035, 1 df). Individuals carrying AA genotype exhibited a decreased risk of developing PD (odds ratio: 0.73; 95% CI: 0.55-0.98, P = 0.035). After stratification by age, individuals over 60 years of age carrying AA genotype demonstrated a further decrease in the risk of developing PD (odds ratio: 0.64; 95% CI: 0.43-0.96, P = 0.030). ChIP-PCR and cDNA over-expression revealed that CCAAT/enhancer-binding protein alpha binds to the ATXN8 proximal promoter to upregulate ATXN8 expression in neuroblastoma SK-N-SH cells. CONCLUSIONS: Our data suggest that ATXN8 -62 G/A polymorphism plays a role in Taiwanese PD susceptibility.

Competition between harmonic cyclotron maser interactions in the terahertz regime.

Cyclotron harmonic interactions are a key physics issue of critical importance to the generation of terahertz radiation via the electron cyclotron maser instability for practical magnetic field strengths. We present an inherent mechanism, as well as a deciding factor, which governs the competition between low- and high-harmonic interactions. Multimode simulations reveal the physical process in which a significant advantage develops for the lower-harmonic interaction, which eventually dominates in the fully nonlinear stage. The results also suggest a start-up scenario for persistent higher-harmonic operation.

Mutations at the mitochondrial DNA polymerase (POLG) locus associated with male infertility.

Human mitochondrial DNA polymerase, encoded by POLG, contains a polyglutamine tract encoded by a CAG microsatellite repeat. Analysis of POLG genotypes in different populations identified an association between absence of the common, ten-repeat allele and male infertility typified by a range of sperm quality defects but excluding azoospermia.

Biomarkers of DNA damage in patients with end-stage renal disease: mitochondrial DNA mutation in hair follicles.

BACKGROUND: DNA damage was noted in patients with end-stage renal disease (ESRD). Mitochondrial DNA (mtDNA) mutations have been proposed as a genomic biomarker in the process of human ageing, degenerative diseases and carcinogenesis. METHODS: Polymerase chain reaction (PCR) techniques were applied to detect mtDNA deletions in hair follicles, an appendage of skin, from 162 patients with ESRD. RESULTS: The incidences of the 4977 bp deletion of mtDNA in hair follicles were found to increase with age in normal control and ESRD patients. As compared with normal subjects, ESRD patients had 3.5, 2.3, 2.7, 2.3 and 1.4 times higher incidences of the 4977 bp deletion of mtDNA in the age groups of 20-30, 31-40, 41-50, 51-60 and 61-70 years, respectively. Moreover, the difference in the proportion of mtDNA with the 4977 bp deletion was statistically significant between ESRD patients and normal subjects >50 years of age. CONCLUSION: We suggest that the 4977 bp deletion of mtDNA in hair follicles may serve as one of the tissue biomarkers of genetic instability of the mitochondrial genome in ESRD patients.

Smoking-associated mitochondrial DNA mutations and lipid peroxidation in human lung tissues.

To investigate the effect of cigarette smoking on mitochondrial DNA (mtDNA) mutation and lipid peroxidation in lung tissues, 152 samples from lung resections were collected. A novel deletion of 4,839 bp of mtDNA was found in 80 (52.6%) of the 152 lung samples. The breakpoints of the 4,839-bp mtDNA deletion were flanked by a nine-nucleotide direct repeat (5'-CATACACAA-3'). The frequency of occurrence and the proportion of the 4,839-bp mtDNA deletion in the lung increased significantly with the smoking index in terms of pack-years (P < 0.05). The incidence and proportion of the 4,839-bp mtDNA deletion in the lung tissues of current smokers were significantly higher than in those of nonsmokers (P < 0.05). In addition, we found that the content of lipid peroxides in the lung tissues of the smokers was significantly higher than in that of nonsmokers, and increased with the smoking index. The average malondialdehyde level in the lung tissues was 12.81 +/- 4.99 micromol/g for subjects with a smoking index of more than 50 pack-yr, and was 5.39 +/- 0.48 micromol/g for nonsmokers (P < 0.05). Multiple regression analysis showed that the smoking index, tissue lipid-peroxide content, and FEV1/FVC ratio were correlated with the proportion of the 4,839-bp mtDNA deletion in the lung. These results suggest that cigarette smoke plays an important role in the increase in mtDNA mutation and lipid peroxidation in the lung tissues of smokers.

Multiple deletions of mitochondrial DNA are associated with the decline of motility and fertility of human spermatozoa.

Sperm motility is one of the major determinants of male fertility and is required for successful fertilization. In a previous study, we demonstrated that the occurrence and accumulation of the 4977 bp deletion of mitochondrial DNA (mtDNA) is associated with diminished fertility and motility of human spermatozoa. The possible relationship between multiple deletions of mtDNA and the decline of fertility and motility in human spermatozoa was further explored in 36 subjects including subfertile and infertile males in this study. Using long-range polymerase chain reaction (PCR), we confirmed the 4977 bp deletion and identified two novel deletions of 7345 and 7599 bp of mtDNA in the spermatozoa with poor motility. We used Percoll gradients to fractionate spermatozoa with differing motility, and then screened for two novel large-scale deletions of the mtDNA. The results showed that the ratio of the deleted mtDNA in the spermatozoa with poor motility and diminished fertility were significantly higher than those in the spermatozoa with good motility and fertility. In addition, we found that the frequencies of the three large-scale deletions in the spermatozoa from patients with primary infertility and oligoasthenozoospermia were higher than those of the fertile males. Our findings suggest that mtDNA deletions may play an important role in some pathophysiological conditions of human spermatozoa.

Serum trace elements, glutathione, copper/zinc superoxide dismutase, and lipid peroxidation in epileptic patients with phenytoin or carbamazepine monotherapy.

Disturbance of metabolism of trace elements and antioxidants are investigated in epileptic patients with long-term therapy of anticonvulsants. One hundred and fifteen subjects including healthy controls, untreated epileptic patients, and phenytoin (PHT)- or carbamazepine (CBZ)-treated epileptic patients were recruited in this study. Serum malondialdehyde was measured as an index of extracellular lipid peroxidation. The levels of serum copper (S-Cu), serum zinc, copper/zinc superoxide dismutase (CuZn-SOD), and reduced glutathione in the serum were monitored simultaneously. The results showed that malondialdehyde, S-Cu, and CuZn-SOD levels in the serum all were significantly increased, but the glutathione level was significantly decreased, in all the epileptic patients with PHT monotherapy compared with those of the controls. However, no significant differences of these parameters in the epileptic patients with CBZ monotherapy were found except for a mild elevation of the activity of serum CuZn-SOD. We conclude that compared with PHT monotherapy, the CBZ monotherapy induced less disturbance in trace element metabolism, antioxidants, and lipid peroxidation in the serum of epileptic patients.

Ageing-associated large-scale deletions of mitochondrial DNA in human hair follicles.

Hair follicles plucked from the bi-temporal region of the scalp of 433 Chinese subjects of different ages were used for the examination of ageing-associated mutations of human mitochondrial DNA (mtDNA). By use of PCR techniques, we detected the 4,977 bp and 7,436 bp deletions of mtDNA in hair follicles from aged individuals. The frequencies of occurrence of both mtDNA deletions were found to increase with age of the subject. Moreover, we employed a semi-quantitative PCR method to determine the proportion of the 4,977 bp deleted mtDNA (dmtDNA) in hair follicles. The results showed that the average proportion of the 4,977 bp dmtDNA in hair follicles were 0.05% +/- 0.01%, 0.00%, 0.55% +/- 0.05%, 0.52% +/- 0.24%, 0.65% +/- 0.17%, 1.33 +/- 0.25%, and 1.89% +/- 0.81% for the subjects in the age groups of 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, and 81-99, respectively. Furthermore, we screened all the subjects harboring the 4,977 bp and/or 7,436 bp deletions for tandem duplications in the D-loop region of mtDNA by PCR with back-to-back primers. The results showed that none of the previously reported tandem duplications were present in all the hair follicles examined. This indicates that tandem duplications do not predispose to large-scale deletions of mtDNA. However, the data suggest that mtDNA deletions occur and accumulate in hair follicles during human ageing. As hair follicles can be easily and non-invasively obtained from the human, we suggest that the aged-dependent accumulation of dmtDNAs in hair follicles may be used for the monitoring of human ageing process.

Phenytoin-mediated oxidative stress in serum of female epileptics: a possible pathogenesis in the fetal hydantoin syndrome.

1. The concentration of serum malondialdehyde (MDA) was measured as the index of lipid peroxidation in female epileptics with phenytoin (PHT) monotherapy. Sera from 20 female epileptics with PHT monotherapy, 12 female epileptics without anticonvulsant therapy and 20 female healthy controls were sampled. The levels of serum copper (S-Cu), serum zinc (S-Zn), copper/zinc superoxide dismutase (CuZn-SOD), and reduced glutathione (GSH) were analyzed as interactive factors of the oxidative stress. 2. For the female epileptics with PHT monotherapy, serum MDA concentration (2.6 +/- 0.7 microM vs control 1.8 +/- 0.6 microM, P < 0.05), CuZn-SOD activity (178.2 +/- 63.5 U/dL vs control 97/1 +/- 36.4 U/dL, P < 0.01), and S-Cu content (126.2 +/- 36.1 micrograms/dL vs control 98.4 +/- 16.7 micrograms/dL, P < 0.05) were significantly increased, but GSH level (27.5 +/- 6.8 microM vs control 32.2 +/- 5.7 microM, P < 0.05) was significantly decreased. The level of serum MDA was associated with the elevation of CuZn-SOD activity (r = 0.54, P < 0.05) and S-Cu content (r = 0.44, P < 0.05) in all the samples collected from epileptics and controls. However, there were no significant differences in all the above parameters between the female epileptics without anticonvulsant therapy and healthy controls. 3. These results indicated that oxidative stress was enhanced in the female epileptics with PHT-monotherapy. Apart from the reactive PHT intermediate, the abnormal metabolism of S-Cu, CuZn-SOD, and GSH was highly involved in the PHT-mediated toxicity. Supplement of GSH, modification of CuZn-SOD enzyme activity and reduction of the absorption of copper may prevent the incidence of fetal hydantoin syndrome during pregnancy.

Smoking-associated mitochondrial DNA mutations in human hair follicles.

The mitochondrial DNA (mtDNA) of hair follicles was used for studying the genotoxicity of smoking-mediated carcinogens. We determined the incidences of the 4,977 bp and 7,436 bp mtDNA deletions, tandem duplication in the D-loop region and the proportion of the 4,977 bp deleted mtDNA (dmtDNA) in the total DNA of hair follicles from 213 male non-smokers and 74 male smokers, respectively. Twenty-three patients with lung cancer were also investigated. We found that the current cigarette smokers had a 3.1 times higher average incidence of the 4,977 bp dmtDNA (RR: 3.1, P < 0.001) as compared with non-smokers, and this mtDNA deletion was especially prevalent in the old heavy smokers. For the smokers of the age above 70, the average incidence of the 4,977 bp dmtDNA was 3.7 times higher in the group with a smoking index of 401-800 (RR: 3.7, P < 0.005) and 3.2 times higher in the group with a smoking index greater than 800 (RR: 3.2, P < 0.005). However, there was no statistically significant relationship between the incidence of the 7,436 bp dmtDNA and the smoking index, although there was a mild increase in the percentage of the 7,436 bp dmtDNA with the increase of the consumption of cigarettes. No tandem duplication of mtDNA in the D-loop region was disclosed in either smokers or non-smokers group. The proportions of the 4,977 bp dmtDNA in hair follicles were found to correlate with age, but did not keep increasing with cigarette consumption except in the group of subjects with a smoking index of less than 400. On the other hand, we found that the average proportion of the 4,977 bp dmtDNA in the hair follicles was 1.201 +/- 0.371% for the patients with lung cancer who had a smoking index greater than 400, while that was only 0.146% for the age-matched healthy smokers with the same smoking index. In conclusion, the high incidence of the 4,977 bp dmtDNA of hair follicles is not only associated with aging but also correlated with the amount of cigarette smoking. A high proportion of the 4,977 bp dmtDNA in the hair follicles may be considered one of the molecular events that are associated with the occurrence of smoking-associated cancers.

Age-related 4,977 bp deletion in human lung mitochondrial DNA.

The accumulation of mitochondrial DNA (mtDNA) mutations has been suggested to be an important contributor to human aging and degenerative diseases. The lung is exposed to ambient air and makes direct contact with the external environment. Numerous potentially noxious agents may damage lung tissues directly or indirectly through free-radical-mediated reactions. In previous studies, we demonstrated an age-dependent increase of mtDNA mutations in various human tissues. We hypothesize that the accumulation of the 4,977 bp (base pairs) deleted mtDNA in human lung tissues is also age-dependent. Using the polymerase chain reaction technique, we determined the incidence of the 4,977 bp-deleted mtDNA in 127 human lung specimens from 34-wk gestation to 79 yr of age. The results showed that 77 lung biopsies (60.6%) contained the 4,977 bp-deleted mtDNA, which started to appear in lung tissues after the fourth decade of life. The incidence apparently increased from 14.3% (one of seven) of the subjects in the 30- to 39-yr age group to 77.8% (two of 27) of the subjects in the 70- to 79-yr age group (p < 0.0001). The mean (+/- SEM) proportion of the 4,977 bp-deleted mtDNA in lung tissues significantly increased from 0.007 +/- 0.007% of the subjects in the 30- to 39-yr age group to 0.833 +/- 0.330% of those in the 70- to 79-yr age group (p < 0.005). Other factors such as sex, pulmonary function indices, and smoking status did not have statistically significant impact on the amount of the deleted mtDNA. These findings suggest that the accumulation of the 4,977 bp-deleted mtDNA is associated with aging human lung.

Simultaneous increase of mitochondrial DNA deletions and lipid peroxidation in human aging.

Human mtDNA is a naked circular double-stranded DNA, which is continually exposed to the matrix that contains high levels of ROS and free radicals. High oxidative stress and a lack of proofreading during mtDNA replication and efficient DNA repair mechanisms in the mitochondria have rendered mtDNA extremely vulnerable to oxidative damage. More than one dozen large-scale deletions in mtDNA have been identified in various tissues of old humans. The 4,977-bp and 7,436-bp deletions are the most prevalent and abundant ones. The onset age of various mtDNA deletions varies greatly with tissues of each individual and type of deletion. In this and previous studies, we have demonstrated with PCR techniques that the frequency of occurrence and the proportion of the 4,977-bp and 7,436-bp deleted mtDNAs are significantly increased with the age of the human. The mtDNA deletions are not detectable in any tissues from young healthy subjects or blood cells from normal individuals of any age, which indicates that the deletions are generated and accumulated only in postmitotic cells upon aging. Moreover, we found that these mtDNA deletions occur more frequently and abundantly in tissues with high energy demand (e.g., muscle) as compared to those with low energy demand. On the other hand, we found that the amount of lipid peroxides measured as malondialdehyde and the activity of manganese-superoxide dismutase in the mitochondria exhibit an age-dependent increase in various human tissues. The lipid peroxide level in muscle was significantly higher than that in the other tissues. Moreover, we found a positive correlation between the proportion of the 4,977-bp deleted mtDNA and lipid peroxide content in the mitochondria of human tissues during aging. Muscle the tissue of high energy demand, was found to be more vulnerable to oxidative damage that lead to most abundant mtDNA deletions and lipid peroxidation among all the tissues examined. Taking these results together, we suggest that the enhanced generation of reactive oxygen species and lipid peroxides in the mitochondria during the aging process occur simultaneously with large-scale deletions and the other types of mutations in mtDNA, which are early molecular events and major contributory factors of human aging.

Stability of an ofloxacin injection in various infusion fluids.

The stability of ofloxacin was evaluated in 10 different infusion fluids under various storage conditions. Solutions of ofloxacin (0.4 mg/mL and 4.0 mg/mL) were prepared in (1) 0.9% sodium chloride injection; (2) 5% dextrose injection; (3) 5% dextrose and 0.9% sodium chloride injection; (4) 5% dextrose and lactated Ringer's injection; (5) 5% sodium bicarbonate injection; (6) Plasma-Lyte 56 and 5% dextrose injection; (7) 5% dextrose, 0.45% sodium chloride, and 0.15% potassium chloride injection; (8) 1/6 M sodium lactate injection; (9) water for injection; and (10) 20% mannitol injection. Each solution was injected into polyvinyl chloride bags and stored at (1) 24 degrees C for 3 days, (2) 5 degrees C for 7 days, (3) 5 degrees C for 14 days, (4) -20 degrees C for 13 weeks and then 5 degrees C for 14 days, or (5) -20 degrees C for 26 weeks and then 5 degrees C, for 14 days. Samples were assayed initially and after storage by high-performance liquid chromatography and examined for visual clarity, pH, turbidity, and particulates. Ofloxacin was stable in all solutions and under all storage conditions. All of the solutions were clear, pH was stable, and particulate-matter counts were acceptable under all storage conditions (except for the 20% mannitol solution, which formed crystals at 5 degrees C and -20 degrees C). An injectable formulation of ofloxacin was stable for at least 3 days at 24 degrees C, 14 days at 5 degrees C, and 26 weeks at -20 degrees C in all tested infusion fluids. Crystals formed in refrigerated or frozen solutions prepared with 20% mannitol injection.

Human follicular fluid stimulates the motility of washed human sperm.

Human follicular fluid (hFF) was collected by laparoscopic oocyte pickup during IVF to evaluate the effect of hFF on human sperm motility with a transmembrane migration method. Freshly ejaculated human sperm were washed with phosphate buffered saline (PBS) and mixed with either PBS or hFF. Amplitude of motility increases were 38% and 72% in washed fertile sperm and washed asthenozoospermic sperm when individual control motility was considered to be 100%. The stimulatory effect of hFF was lost when preheated at 100 degrees C for 30 minutes. hFF collected from mature follicles stimulated sperm motility better than that collected from intermediate or immature follicles. hFF did not stimulate the motility of unwashed sperm in freshly ejaculated human semen. A heat labile factor(s) in hFF may stimulate the motility of washed human sperm. Whether this factor could be used to improve the success rate of IVF and artificial insemination awaits further investigation.

Roles of bacteriophage T4 gene 5 and gene s products in cell lysis.

Previous studies indicated that (i) T4 gene s product (gps) protects infected cells from superinfection lysis from without, (ii) the absence of gps in infected cells also leads to lysis from within even when T4 e lysozyme is absent, (iii) T4 gene 5 product (gp5), a polypeptide of the virion baseplate, may be responsible for inducing lysis from without, and (iv) altered gp5 of the T4 mutant 5ts1 can replace e lysozyme to cause lysis from within. Results of this study showed that (i) wild-type gp5 in infected cells lacking e lysozyme was responsible for lysis from within in the absence of gps, and (ii) gps did not protect infected cells from superinfection lysis from without by 5ts1 phage. We prpose that gps normally prevents functional expression of wild-type gp5 activity from either side of the cell wall, whereas the 5ts1 form of gp5 is insensitive to the gps barrier and induces lysis from either side of the cell wall.

Baseplate protein of bacteriophage T4 with both structural and lytic functions.

Analyses of a new bacteriophage T4 mutant that permits lysis of infected cells in the absence of e lysozyme showed that the strain carried a suppressor mutation in gene 5, a gene whose polypeptide product (gp5) is an integral component of the virion baseplate. Indirect experiments indicated that cell lysis was caused by the lytic action of mutant gp5. With regard to the physiological role of normal gp5, we speculate that it functions in the initiation of infection by catalyzing local cell wall digestion to facilitate penetration of the tail tube through the cell envelope. The proposed lytic activity of gp5 may also be responsible for the well-known phenomenon of lysis from without observed with T4.

U-G-A suppressor of bacteriophage T4 associated with arginine transfer RNA.

A U-G-A suppressor of bacteriophage T4, designated psu4+op, has been isolated and characterized. The transfer RNA species previously shown to have an anticodon sequence complementary to arginine codons is affected by the psu4+op mutation. Wild type and psu4+op arginine tRNAs have the same sequence except for their anticodons, where U-C-U in the wild type species is mutated to U-C-A in the psu4+op species. This mutation is believed to confer U-G-A suppressor activity on the psu4+op arginine tRNA.