Characteristics of long working hours and subsequent psychological and physical responses: JNIOSH cohort study.

OBJECTIVES: This study aimed to examine the prospective association among objectively measured average working hours (AWHs), frequency of long working hours (FLWHs; defined as ≥205 working hours/month (≥45 hours/week)) for 6 months, and workers' self-reported psychological and physical health. METHODS: The study included 15 143 workers from 5 Japanese companies. We collected monthly attendance records over 6 months before distributing a questionnaire survey on psychological/physical stress responses and work-related demographics. We then evaluated the associations of those attendance records with psychological/physical measures using analysis of covariance adjusted for sex, age, employment, job type, working conditions, work site and experience of emergency state due to COVID-19. RESULTS: Irritability, anxiety and depression were significantly greater at ≥180 hours (≥45 hours/week), and fatigue and lack of vigour were greater at ≥205 hours than those of the normal working-hour group (140-180 hours/month [35-45 hours/week]). Psychological indices increased significantly with FLWH, with ≥3 times for irritability, depression and fatigue; ≥2 times for lack of vigour; and ≥1 time for anxiety when compared with no long working hours. No significant associations were observed between AWH or FLWH and physical stress responses. CONCLUSIONS: Longer AWH was associated with higher levels of psychological stress responses. The effects of FLWH in the past 6 months varied among the psychological stress responses and did not occur for physical complaints. Under circumstances requiring long hours, workers' mental health should be protected through minimising the frequency of long work hours.

Health problems associated with single, multiple, and the frequency of months of objectively measured long working hours: a cohort study by the National Institute of Occupational Safety and Health, Japan.

PURPOSE: We aimed to examine the prospective associations of monthly working hours measured in a month, the 6-month averaged hours, and the frequency of long working hours (≥ 205 h/month) during the past 6 months with health indicators. METHODS: This study included 6,806 Japanese company workers (response rate = 86.6%). Data on the workers' monthly attendance during the second half of fiscal year 2016 and annual health checkups in fiscal years 2016 and 2017 were collected. We evaluated the association of the above three types of monthly working hours with subsequent health checkup data in fiscal year 2017. We adjusted for the corresponding data in fiscal year 2016. RESULTS: Multivariate logistic regression analyses revealed significant associations between monthly working hours and workers' systolic and diastolic blood pressure as well as aspartate aminotransferase, alanine aminotransferase, low-density lipoprotein cholesterol (LDL), and triglyceride levels. However, the associations were not consistent between months. The average monthly working hours were significantly associated with higher LDL levels for the 220-240 h/mo group (OR: 1.49, 95%CI: 1.07-2.08) and lower triglyceride levels for the < 140 h/mo group (OR: 0.15, 95%CI: 0.03-0.77), compared to the 140-180 h/mo group. The frequency of long working hours was significantly associated with higher LDL levels. CONCLUSIONS: Working hours over several months produced various associations with health indicators compared to those measured in a single month. Our present data suggest that the effects of average or frequency of long working hours during the past 6 months are likely to appear in LDL levels.

Ubiquitinated sirtuin 1 (SIRT1) function is modulated during DNA damage-induced cell death and survival.

Downstream signaling of physiological and pathological cell responses depends on post-translational modification such as ubiquitination. The mechanisms regulating downstream DNA damage response (DDR) signaling are not completely elucidated. Sirtuin 1 (SIRT1), the founding member of Class III histone deacetylases, regulates multiple steps in DDR and is closely associated with many physiological and pathological processes. However, the role of post-translational modification or ubiquitination of SIRT1 during DDR is unclear. We show that SIRT1 is dynamically and distinctly ubiquitinated in response to DNA damage. SIRT1 was ubiquitinated by the MDM2 E3 ligase in vitro and in vivo. SIRT1 ubiquitination under normal conditions had no effect on its enzymatic activity or rate of degradation; hypo-ubiquitination, however, reduced SIRT1 nuclear localization. Ubiquitination of SIRT1 affected its function in cell death and survival in response to DNA damage. Our results suggest that ubiquitination is required for SIRT1 function during DDR.

Inhibition of Crm1-p53 interaction and nuclear export of p53 by poly(ADP-ribosyl)ation.

Poly(ADP-ribose) polymerase 1 (PARP-1) and p53 are two key proteins in the DNA-damage response. Although PARP-1 is known to poly(ADP-ribosyl)ate p53, the role of this modification remains elusive. Here, we identify the major poly(ADP-ribosyl)ated sites of p53 by PARP-1 and find that PARP-1-mediated poly(ADP-ribosyl)ation blocks the interaction between p53 and the nuclear export receptor Crm1, resulting in nuclear accumulation of p53. These findings molecularly link PARP-1 and p53 in the DNA-damage response, providing the mechanism for how p53 accumulates in the nucleus in response to DNA damage. PARP-1 becomes super-activated by binding to damaged DNA, which in turn poly(ADP-ribosyl)ates p53. The nuclear export machinery is unable to target poly(ADP-ribosyl)ated p53, promoting accumulation of p53 in the nucleus where p53 exerts its transactivational function.

Comparison of body mass index, waist circumference, and waist-to-height ratio for predicting the clustering of cardiometabolic risk factors by age in Japanese workers--Japan Epidemiology Collaboration on Occupational Health study.

BACKGROUND: Waist-to-height ratio (WHtR) has been suggested as a better screening tool than body mass index (BMI) and waist circumference (WC) for assessing cardiometabolic risk. However, most previous studies did not consider age. METHODS AND RESULTS: Participants were 45,618 men and 8,092 women aged 15-84 years who received periodic health checkups in 9 companies in Japan. Clustering of cardiometabolic risk factors was defined by the existence of 2 or more of high blood pressure, hyperglycemia, and dyslipidemia. In both men and women, unadjusted area under the curve (AUC) of the receiver-operating characteristic curve for WHtR in detecting the clustering of cardiometabolic risk factors was significantly higher than that for either BMI or WC; the AUCs for WHtR, BMI, and WC, respectively, were 0.734, 0.705, and 0.717 in men and 0.782, 0.762, and 0.755 in women. After adjustment for age, however, such differences were not observed; the corresponding values were 0.702, 0.701, and 0.696 in men. In women, the age-adjusted AUC for BMI was slightly higher than for other indices (WHtR, 0.721; BMI, 0.726; WC, 0.707). CONCLUSIONS: The screening performance of WHtR for detecting the clustering cardiometabolic risk factors was not superior to that of BMI.

Inactivation of E2F3 results in centrosome amplification.

The E2F family of transcription factors is critical for the control of cell cycle progression. We now show that the specific inactivation of E2F3 in mouse embryo fibroblasts (MEFs) results in a disruption of the centrosome duplication cycle. Loss of E2F3, but not E2F1, E2F2, E2F4, or E2F5 results in unregulated cyclin E-dependent kinase activity, defects in nucleophosmin B association with centrosomes, and premature centriole separation and duplication. Consequently, this defect leads to centrosome amplification, mitotic spindle defects, and aneuploidy. Our findings implicate the E2F3 transcription factor as an important link that orchestrates DNA and centrosome duplication cycles, ensuring the faithful transmission of genetic material to daughter cells.

Study profile: protocol outline and study perspectives of the cohort by the National Institute of Occupational Safety and Health, Japan (JNIOSH cohort).

How work burden affects physical and mental health has already been studied extensively; however, many issues have remained unexamined. In 2017, we commenced a prospective cohort study of workers at companies in Japan, with a follow-up period of 5-10 years, in order to investigate the current situation of overwork-related health outcomes. From 2017 to 2020, a target population of 150,000 workers across 8 companies was identified. Of these, almost 40,000 workers agreed to participate in the baseline survey. Data on working hours, medical check-up measurements, occupational stress levels, and lifestyle habits were collected. The average age of the participants at baseline was 39.2 ± 11.7 years; 73.1% were men, and 87.7% were regular employees. The most common working hours by self-reported was 41-50 hours per week during normal season, and it increased to more than 50 hours during busy season. Furthermore, more than half of the participants reportedly experienced a form of sleep problem, and the percentage of those who experienced nonrestorative sleep was particularly high.

Objective and Subjective Working Hours and Their Roles on Workers' Health among Japanese Employees.

Table 3 of the above paper appeared incorrectly in print. Percentage figures on the table were inadvertently listed as negative values. These errors were corrected in online versions of this paper, as shown below.

Objective and subjective working hours and their roles on workers' health among Japanese employees.

This study investigated the correlation between objective and subjective working hours (OWH and SWH, respectively) and their relation to the workers' health. The study included 6,806 workers of a Japanese company (response rate=86.6%). OWH were collected as the monthly data during fiscal year 2017 from the company record. SWH were self-reported as the weekly data during the past month in November 2017. Both OWH and SWH corresponded to the same period of one month (October 2017). Additionally, the data for the annual health checkup in fiscal year 2017 and self-reported mental health in November 2017 were collected. The results indicated that the longer OWH was related to more underestimation of SWH. The analyses of covariance adjusted for the selected variables showed that irrespective of OWH or SWH, significant relationships were found for stress responses but not for body mass index, aspartate and alanine aminotransferase, fasting blood glucose, hemoglobin A1c, high-density lipoprotein cholesterol, or triglyceride. However, significant relationships with only OWH were noted for systolic and diastolic blood pressure, low-density lipoprotein cholesterol, gamma-glutamyl transpeptidase, and positive work-related state of mind. The present findings show that SWH should be used carefully when assessing the health effects of long working hours.

P53, cyclin-dependent kinase and abnormal amplification of centrosomes.

Centrosomes play a critical role in formation of bipolar mitotic spindles, an essential event for accurate chromosome segregation into daughter cells. Numeral abnormalities of centrosomes (centrosome amplification) occur frequently in cancers, and are considered to be the major cause of chromosome instability, which accelerates acquisition of malignant phenotypes during tumor progression. Loss or mutational inactivation of p53 tumor suppressor protein, one of the most common mutations found in cancers, results in a high frequency of centrosome amplification in part via allowing the activation of the cyclin-dependent kinase (CDK) 2-cyclin E (as well as CDK2-cyclin A) which is a key factor for the initiation of centrosome duplication. In this review, the role of centrosome amplification in tumor progression, and mechanistic view of how centrosomes are amplified in cells through focusing on loss of p53 and aberrant activities of CDK2-cyclins will be discussed.

Interaction between ROCK II and nucleophosmin/B23 in the regulation of centrosome duplication.

Nucleophosmin (NPM)/B23 has been implicated in the regulation of centrosome duplication. NPM/B23 localizes between two centrioles in the unduplicated centrosome. Upon phosphorylation on Thr(199) by cyclin-dependent kinase 2 (CDK2)/cyclin E, the majority of centrosomal NPM/B23 dissociates from centrosomes, but some NPM/B23 phosphorylated on Thr(199) remains at centrosomes. It has been shown that Thr(199) phosphorylation of NPM/B23 is critical for the physical separation of the paired centrioles, an initial event of the centrosome duplication process. Here, we identified ROCK II kinase, an effector of Rho small GTPase, as a protein that localizes to centrosomes and physically interacts with NPM/B23. Expression of the constitutively active form of ROCK II promotes centrosome duplication, while down-regulation of ROCK II expression results in the suppression of centrosome duplication, especially delaying the initiation of centrosome duplication during the cell cycle. Moreover, ROCK II regulates centrosome duplication in its kinase and centrosome localization activity-dependent manner. We further found that ROCK II kinase activity is significantly enhanced by binding to NPM/B23 and that NPM/B23 acquires a higher binding affinity to ROCK II upon phosphorylation on Thr(199). Moreover, physical interaction between ROCK II and NPM/B23 in vivo occurs in association with CDK2/cyclin E activation and the emergence of Thr(199)-phosphorylated NPM/B23. All these findings point to ROCK II as the effector of the CDK2/cyclin E-NPM/B23 pathway in the regulation of centrosome duplication.

Transcriptional control of BubR1 by p53 and suppression of centrosome amplification by BubR1.

Elimination of the regulatory mechanism underlying numeral homeostasis of centrosomes, as seen in cells lacking p53, results in abnormal amplification of centrosomes, which increases the frequency of chromosome segregation errors, and thus contributes to the chromosome instability frequently observed in cancer cells. We have previously reported that p53(-/-) mouse cells in prolonged culture undergo genomic convergence similar to that observed during tumor progression; early-passage p53(-/-) cells are karyotypically heterogeneous due to extensive chromosome instability associated with centrosome amplification, while late-passage p53(-/-) cells are aneuploid yet karyotypically homogeneous and chromosomally stable. Moreover, they contain numerically normal centrosomes. Through the microarray analysis of early- and late-passage p53(-/-) cells, we identified the BubR1 spindle checkpoint protein, which plays a critical role in suppression of centrosome amplification and stabilization of chromosomes in late-passage p53(-/-) cells. Up-regulation of BubR1 augments the checkpoint function, which effectively senses the spindle/chromosome aberrations associated with centrosome amplification. We further found that BubR1 transcription is largely controlled by p53. In early-passage p53(-/-) cells, BubR1 expression is low and the checkpoint function in response to microtubule toxin is considerably compromised. In late-passage cells, however, regaining of BubR1 expression restores the checkpoint function to mitotic aberrations caused by microtubule toxin. Our studies demonstrate the molecular aspect of genomic convergence in cultured cells, providing critical information for understanding the stepwise progression of tumors.

Suppression of centrosome amplification after DNA damage depends on p27 accumulation.

The centrosome plays a fundamental role in cell division, cell polarity, and cell cycle progression. Centrosome duplication is mainly controlled by cyclin-dependent kinase 2 (CDK2)/cyclin E and cyclin A complexes, which are inhibited by the CDK inhibitors p21Cip1 and p27Kip1. It is thought that abnormal activation of CDK2 induces centrosome amplification that is frequently observed in a wide range of aggressive tumors. We previously reported that overexpression of the oncogene MYCN leads to centrosome amplification after DNA damage in neuroblastoma cells. We here show that centrosome amplification after gamma-irradiation was caused by suppression of p27 expression in MYCN-overexpressing cells. We further show that p27-/- and p27+/- mouse embryonic fibroblasts and p27-silenced human cells exhibited a significant increase in centrosome amplification after DNA damage. Moreover, abnormal mitotic cells with amplified centrosomes were frequently observed in p27-silenced cells. In response to DNA damage, the level of p27 gradually increased in normal cells independently of the ataxia telangiectasia mutated/p53 pathway, whereas Skp2, an F-box protein component of an SCF ubiquitin ligase complex that targets p27, was reduced. Additionally, p27 levels in MYCN-overexpressing cells were restored by treatment with Skp2 small interfering RNA, indicating that down-regulation of p27 by MYCN was due to high expression of Skp2. These results suggest that the accumulation of p27 after DNA damage is required for suppression of centrosome amplification, thereby preventing chromosomal instability.

Correction: Development of Risk Score for Predicting 3-Year Incidence of Type 2 Diabetes: Japan Epidemiology Collaboration on Occupational Health Study.

[This corrects the article DOI: 10.1371/journal.pone.0142779.].

Involvement of poly(ADP-Ribose) polymerase 1 and poly(ADP-Ribosyl)ation in regulation of centrosome function.

The regulatory mechanism of centrosome function is crucial to the accurate transmission of chromosomes to the daughter cells in mitosis. Recent findings on the posttranslational modifications of many centrosomal proteins led us to speculate that these modifications might be involved in centrosome behavior. Poly(ADP-ribose) polymerase 1 (PARP-1) catalyzes poly(ADP-ribosyl)ation to various proteins. We show here that PARP-1 localizes to centrosomes and catalyzes poly(ADP-ribosyl)ation of centrosomal proteins. Moreover, centrosome hyperamplification is frequently observed with PARP inhibitor, as well as in PARP-1-null cells. Thus, it is possible that chromosomal instability known in PARP-1-null cells can be attributed to the centrosomal dysfunction. P53 tumor suppressor protein has been also shown to be localized at centrosomes and to be involved in the regulation of centrosome duplication and monitoring of the chromosomal stability. We found that centrosomal p53 is poly(ADP-ribosyl)ated in vivo and centrosomal PARP-1 directly catalyzes poly(ADP-ribosyl)ation of p53 in vitro. These results indicate that PARP-1 and PARP-1-mediated poly(ADP-ribosyl)ation of centrosomal proteins are involved in the regulation of centrosome function.

Cell cycle arrest and apoptosis induced by human Polo-like kinase 3 is mediated through perturbation of microtubule integrity.

Human Polo-like kinase 3 (Plk3, previously termed Prk or Fnk) is involved in regulation of cell cycle progression through the M phase (B. Ouyang, H. Pan, L. Lu, J. Li, P. Stambrook, B. Li, and W. Dai, J. Biol. Chem. 272:28646-28651, 1997). Here we report that in most interphase cells endogenous Plk3 was predominantly localized around the nuclear membrane. Double labeling with Plk3 and gamma-tubulin, the latter a major component of pericentriole materials, revealed that Plk3 was closely associated with centrosomes and that its localization to centrosomes was dependent on the integrity of microtubules. Throughout mitosis, Plk3 appeared to be localized to mitotic apparatus such as spindle poles and mitotic spindles. During telophase, a significant amount of Plk3 was also detected in the midbody. Ectopic expression of Plk3 mutants dramatically changed cell morphology primarily due to their effects on microtubule dynamics. Expression of a constitutively active Plk3 (Plk3-A) resulted in rapid cell shrinkage, which led to formation of cells with an elongated, unsevered, and taxol-sensitive midbody. In contrast, cells expressing a kinase-defective Plk3 (Plk3(K52R)) mutant exhibited extended, deformed cytoplasmic structures, the phenotype of which was somewhat refractory to taxol treatment. Expression of both Plk3-A and Plk3(K52R) induced apparent G(2)/M arrest followed by apoptosis, although the kinase-defective mutant was less effective. Taken together, our studies strongly suggest that Plk3 plays an important role in the regulation of microtubule dynamics and centrosomal function in the cell and that deregulated expression of Plk3 results in cell cycle arrest and apoptosis.

Involvement of Crm1 in hepatitis B virus X protein-induced aberrant centriole replication and abnormal mitotic spindles.

Hepatitis B virus (HBV) includes an X gene (HBx gene) that plays a critical role in liver carcinogenesis. Because centrosome abnormalities are associated with genomic instability in most human cancer cells, we examined the effect of HBx on centrosomes. We found that HBx induced supernumerary centrosomes and multipolar spindles. This effect was independent of mutations in the p21 gene. Furthermore, the ability of HBV to induce supernumerary centrosomes was dependent on the presence of physiological HBx expression. We recently showed that HBx induces cytoplasmic sequestration of Crm1, a nuclear export receptor that binds to Ran GTPase, thereby inducing nuclear localization of NF-kappaB. Consistently, supernumerary centrosomes were observed in cells treated with a Crm1-specific inhibitor but not with an HBx mutant that lacked the ability to sequester Crm1 in the cytoplasm. Moreover, a fraction of Crm1 was found to be localized at the centrosomes. Immunocytochemical and ultrastructural examination of these supernumerary centrosomes revealed that inactivation of Crm1 was associated with abnormal centrioles. The presence of more than two centrosomes led to an increased frequency of defective mitoses and chromosome transmission errors. Based on this evidence, we suggest that Crm1 is actively involved in maintaining centrosome integrity and that HBx disrupts this process by inactivating Crm1 and thus contributes to HBV-mediated carcinogenesis.

Sickness absence in relation to psychosocial work factors among daytime workers in an electric equipment manufacturing company.

Associations between psychosocial work factors and sickness absence were investigated in a cross-sectional study of 833 daytime workers. Participants completed a questionnaire regarding psychosocial work factors using the US National Institute for Occupational Safety and Health Generic Job Stress Questionnaire (job control, quantitative workload, cognitive demands, variance in workload, intragroup conflict, intergroup conflict, supervisor support, coworker support, family support, job satisfaction and depressive symptoms) and the number of days of sickness absence within the previous year. Multivariate analyses of covariance with age and occupation as covariates (MANCOVA) were used to test the relationships between psychosocial work factors and sickness absence stratified by sex. In men, the age-adjusted MANCOVA showed that, quantitative workload was highest in the 0.5-4.5 d of sickness absence group (p<0.001). However, the levels of stress reactions (job satisfaction and depressive symptoms) in this group were almost identical to the levels recorded in the no sickness absence group. In contrast, low levels of job control (p<0.01), supervisor support (p<0.05), and job satisfaction (p<0.01) and higher symptoms of depression (p<0.001) were associated with 5 d or more sickness absence. In women, only high job satisfaction was associated with 5 d or more sickness absence (p<0.10). This study suggests that appropriate use of sickness absence at times of being exposed to high quantitative workload may help male workers to recover from stressful situations.

Liver-specific pRB loss results in ectopic cell cycle entry and aberrant ploidy.

The liver exhibits an exquisitely controlled cell cycle, wherein hepatocytes are maintained in quiescence until stimulated to proliferate. The retinoblastoma tumor suppressor, pRB, plays a central role in proliferative control by inhibiting inappropriate cell cycle entry. In many cases, liver cancer arises due to aberrant cycles of proliferation, and correspondingly, pRB is functionally inactivated in the majority of hepatocellular carcinomas. Therefore, to determine how pRB loss may provide conditions permissive for deregulated hepatocyte proliferation, we investigated the consequence of somatic pRB inactivation in murine liver. We show that liver-specific pRB loss results in E2F target gene deregulation and elevated cell cycle progression during post-natal growth. However, in adult livers, E2F targets are repressed and hepatocytes become quiescent independent of pRB, suggesting that other factors may compensate for pRB loss. Therefore, to probe the consequences of acute pRB inactivation in livers of adult mice, we gave adenoviral-Cre by i.v. injection. We show that acute pRB loss is sufficient to elicit E2F target gene expression and cell cycle entry in adult liver, demonstrating a critical role for pRB in maintaining hepatocyte quiescence. Finally, we show that liver-specific pRB loss results in the development of nuclear pleomorphism associated with elevated ploidy that is evident in adult mice harboring both acute and chronic pRB loss. Together, these results show the crucial role played by pRB in maintaining hepatocyte quiescence and ploidy in adult liver in vivo and underscore the critical importance of delineating the consequences of acute pRB loss in adult animals.

Thr199 phosphorylation targets nucleophosmin to nuclear speckles and represses pre-mRNA processing.

Nucleophosmin (NPM) is a multifunctional phosphoprotein, being involved in ribosome assembly, pre-ribosomal RNA processing, DNA duplication, nucleocytoplasmic protein trafficking, and centrosome duplication. NPM is phosphorylated by several kinases, including nuclear kinase II, casein kinase 2, Polo-like kinase 1 and cyclin-dependent kinases (CDK1 and 2), and these phosphorylations modulate the activity and function of NPM. We have previously identified Thr(199) as the major phosphorylation site of NPM mediated by CDK2/cyclin E (and A), and this phosphorylation is involved in the regulation of centrosome duplication. In this study, we further examined the effect of CDK2-mediated phosphorylation of NPM by using the antibody that specifically recognizes NPM phosphorylated on Thr(199). We found that the phospho-Thr(199) NPM localized to dynamic sub-nuclear structures known as nuclear speckles, which are believed to be the sites of storage and/or assembly of pre-mRNA splicing factors. Phosphorylation on Thr(199) by CDK2/cyclin E (and A) targets NPM to nuclear speckles, and enhances the RNA-binding activity of NPM. Moreover, phospho-Thr(199) NPM, but not unphosphorylated NPM, effectively represses pre-mRNA splicing. These findings indicate the involvement of NPM in the regulation of pre-mRNA processing, and its activity is controlled by CDK2-mediated phosphorylation on Thr(199).