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The child welfare workforce faces looming staffing shortages complicated by the large number of workers approaching retirement. Strategies that mitigate the loss of talented older workers to retirement represent a partial solution. However, child welfare research to date has not examined whether or how older workers might differ from younger workers in terms of retention-related issues. To address this gap, this study utilizes an integration of two theoretical perspectives -- organizational climate theory and the life course perspective -- as a guiding framework. Data from a sample of 432 public child welfare workers were analyzed in terms of moderating effects of age on the relationship between individual and organizational factors on work and job withdrawal. Results indicate that age moderates the relationship between perceived stress and work withdrawal (i.e., disengagement from work while remaining in the job) and between organizational commitment and job withdrawal (i.e., leaving the job entirely). Practice and research implications are discussed for retention and delaying retirement of talented and engaged mature workers interested in remaining employed. [ABSTRACT FROM AUTHOR]

This research examined the understudied field practicum experiences of macro-oriented (management and community practice) social work graduate students in macro practice settings. MANOVA results suggest that, although macro and clinical practice student s (n = 477) were similar in many ways, macro practice students, regardless of age, experience, or program status, reported less positive field placement experiences than clinical practice students. Implications and recommendations are discussed for macro-level social work education, the structure of management and community practice fieldwork, and supervision in macro-oriented practicum. [ABSTRACT FROM AUTHOR]

The purpose of the study was to explore 115 human services manager's responses to current challenges, and determine whether or not managers engaged in the kind of administrative practice that has been advocated for in the human service management literature. Interview data revealed that the primary concerns among managers appeared to be related to resource development and organizational climate, and the solutions included staff development, resource development through fundraising and grant writing, staff recruitment, and some strategic planning. Binary logistic regression was used to predict the occurrence of identified challenges and solutions based on agency type, size, and location. Overall, the findings reflect a disparity between current management practices and expectations for more visionary and innovative responses. Often, solutions did not match with the identified challenges. The findings indicate that human service managers have to move beyond solving systematic problerns with symptomatic solutions, and recommendations were discussed. [ABSTRACT FROM AUTHOR]

In an era of 'doing more with less,' social service organizations expect workers to engage in 'organizational citizenship' behaviors-extra-role activities that are not formally required by the job-that benefit both the organization and clients. Organizational citizenship is considered critical in for-profit organizations because it is related to organizational effectiveness and quality service, but it has not been examined in social service organizations. This pilot study explored organizational citizenship behavior by surveying 120 randomly selected child and family service employees. Results showed that workers' reports of organizational support, developmental experiences, quality work performance and professional education were positively related to workers' citizenship behavior. More research is needed, including examining the link between organizational citizenship and service quality in social service agencies. [ABSTRACT FROM AUTHOR]

Background: Outbreaks linked to inadequate endoscope drying have infected numerous patients, and current standards and guidelines recommend at least 10 minutes of forced air for drying channels. This study evaluated a new forced-air drying system (FADS) for endoscopes., Methods: Drying was assessed using droplet detection cards; visual inspection of air/water connectors, suction connectors, and distal ends; and borescope examinations of endoscope interiors. Assessments were performed after automated endoscope reprocessor (AER) alcohol flush and air purge cycles and after 10-minute FADS cycles., Results: Researchers evaluated drying during encounters with 22 gastroscopes and 20 colonoscopes. After default AER alcohol and air purge cycles, 100% (42/42) of endoscopes were still wet. Substantial fluid emerged from distal ends during the first 15 seconds of the FADS cycle, and droplets also emerged from air/water and suction connectors. Following FADS cycle completion, 100% (42/42) were dry, with no retained fluid detected by any of the assessment methods., Conclusions: Multiple endoscope ports and channels remained wet after AER cycles intended to aid in drying but were dry after the FADS cycle. This study reinforced the need to evaluate the effectiveness of current drying practices and illustrated the use of practical tools in a real-world setting., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: Borescope examinations of endoscope channels are commonly described in literature, but no studies on surgical instrument lumen inspection have been published recently. Inadequately processed surgical instruments have been implicated in patient infections. This study assessed the utility of borescopes for inspecting surgical instruments. Methods: The study team inspected and photographed sterilized, patient-ready arthroscopic shaver handpieces and suction tips using a tablet camera and borescopes to characterize internal anatomy, defects found in lumens, and the impact of recleaning on debris or residues. Results: Ten suctions and eight shavers were inspected. All suctions had internal ridges and suction holes that were perpendicular to the lumen. All shavers had visible ridges, elbows, and lever mechanisms inside lumens. Of the 18 instruments, 16 (88%) had internal features that appeared rough or jagged and 17 (94%) had visible debris or discoloration in the lumens. Recleaning efforts generally were effective for suctions, but multiple rounds of recleaning with enhanced steps were less effective for shavers, which were replaced. Researchers documented retained soil and brush bristles in several new shavers despite following manufacturer instructions for cleaning and found visible damage and discoloration within five uses. Discussion: This study demonstrated the value of borescope examinations for surgical instrument lumens. Visual inspections identified anatomical features that could influence cleaning effectiveness and detected residual soil, discoloration, and debris in most instruments. The findings suggested that manufacturer cleaning instructions were insufficient and additional cleaning was not always effective. In response, the site's multidisciplinary team strengthened risk assessment protocols and enhanced their cleaning practices.

Background: Health care is shifting toward minimally invasive procedures requiring increasingly complex instruments and sophisticated processing technologies. Effective training methods are needed to ensure sterile processing professionals acquire and retain essential skills. This study aimed to develop and evaluate a new training model that supports mastery and retention of complex key skills., Methods: The model was pilot-tested with training focused on visual inspection of endoscopes. Pre- and post-training tests were administered to enhance learning during a face-to-face workshop that interspersed lectures and hands-on practice, followed by structured homework, and an online booster session. Surveys assessed satisfaction and confidence levels., Results: Mean test scores for nine certified sterile processing employees increased significantly following the workshop (41% vs 84%, P < .001). After the workshop, all trainees identified actionable visible defects on patient-ready endoscopes in their facilities. Test scores remained high after 2 months (90%), and trainees reported higher technical confidence and satisfaction levels after training., Conclusions: This study demonstrated effectiveness and clinical relevance of a new, evidence-based model for training sterile processing professionals that incorporated pretesting, lectures, hands-on practice, a training booster, and post-testing to enhance learning. This model may be applicable to other complex skills necessary for infection prevention and patient safety., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Infections have been linked to damaged or contaminated endoscopes with visible defects. Endoscope processing standards and guidelines state endoscopes should be visually inspected every time they are used. This study evaluated a new visual inspection program using magnification and borescopes in an endoscopy department that had not previously utilized these tools., Methods: Site personnel were given visual inspection tools and training before systematically examining fully processed endoscopes twice during a 2-month period. A risk assessment protocol was used to determine whether endoscopes required recleaning, repair, or other action. Findings were documented using log sheets, photographs, and videotapes., Results: Visible damage and residue or debris were observed in 100% of 25 endoscopes at both assessments, and 76% required repair. Defects at baseline included scratches (88%); channel shredding or peeling (80%); adhesive band disintegration (80%); residual soil or debris (white 84%; black 68%; brown 40%; yellow/green 36%; and orange/red 8%); retained fluid (52%); and dents (40%). Findings were similar at follow-up., Discussion/conclusions: Visual inspection with magnification and borescopes identified actionable defects that could interfere with processing effectiveness in 100% of endoscopes. Infection preventionists have a critical role to play in supporting processing personnel now that standards, guidelines, and manufacturer instructions recommend enhanced visual inspection of every endoscope, every time., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Sterile processing personnel routinely decontaminate medical devices that are heavily soiled with blood, tissue, and secretions. Contamination may spread throughout processing areas, potentially exposing personnel and patient-ready devices, especially when there is insufficient separation between the dirty and clean areas., Objective: This study aimed to identify activities that generate splash, determine how far droplets travel during manual cleaning, characterize the impact of practices on splash generation, and assess effectiveness of personal protective equipment (PPE) at preventing splash exposure to technicians and visitors in the decontamination unit., Methods: Moisture-detection paper was affixed to PPE and environmental surfaces in a new processing department designed to optimize workflow and prevent cross-contamination. Droplet generation and dispersal were assessed during manual cleaning of a colonoscope and a transvaginal ultrasound probe., Results: Splash was generated by most activities and droplets were detected up to 7.25 feet away. Transporting wet endoscopes dispersed droplets on a 15-foot path from the sink to the automated endoscope reprocessor. Extensive droplets were detected on PPE worn by technicians at the sink and observers 3-4 feet away., Conclusions: Manual cleaning of devices generated substantial splash, drenching technicians and the environment with droplets that traveled more than 7 feet. Engineering controls and better PPE are needed to reduce personnel exposure and risks associated with the potential dispersal of contaminated fluids throughout the facility., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Flexible ureteroscopes are essential devices, and reusable models require meticulous cleaning and sterilization between procedures. Reprocessing is complex, time-consuming, and difficult due to the instrument's small size, fragility, and internal channel. The Food and Drug Administration recently raised concerns about the effectiveness of ureteroscope reprocessing. Due to inadequate reprocessing and maintenance, contaminated and damaged ureteroscopes pose risks to patient safety. This review describes ureteroscope reprocessing methods and summarizes evidence on reprocessing effectiveness and documented outcomes associated with the use of damaged or inadequately cleaned and sterilized ureteroscopes., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Personnel working in sterile processing or endoscope reprocessing departments are at high risk of exposure to tissue, blood, and patient fluids when decontaminating reusable medical instruments and equipment. The effectiveness of protective measures for reprocessing personnel has not yet been systematically evaluated in real-world settings., Objective: This pilot project aimed to identify reprocessing activities that generate splashes, determine how far droplets can travel in decontamination areas, and assess personal protective equipment exposure during routine activities., Methods: Moisture-detection paper was affixed to environmental surfaces and personal protective equipment in a sterile processing department. Droplet dispersal was assessed after personnel simulated performance of routine reprocessing tasks., Results: Visible droplets were generated during every reprocessing activity except running the sonication sink. Droplets traveled at least 3 feet when filling a sink, brushing a ureteroscope, and using a power sprayer to rinse a basin. Some activities dispersed droplets up to 5 feet from the sink. Personal protective equipment was splashed during most activities and did not prevent skin exposure even when properly donned and doffed., Conclusion: This hypothesis-generating pilot project found that routine reprocessing activities generated substantial splashing, and currently recommended personal protective equipment did not adequately protect sterile processing personnel from exposure., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Prostate cancer is the second most common type of cancer and the second leading cause of cancer death in American men. RAD9 stabilizes the genome, but prostate cancer cells and tumors often have high quantities of the protein. Reduction of RAD9 level within prostate cancer cells decreases tumorigenicity of nude mouse xenographs and metastasis phenotypes in culture, indicating that RAD9 overproduction is essential for the disease. In prostate cancer DU145 cells, CpG hypermethylation in a transcription suppressor site of RAD9 intron 2 causes high-level gene expression. Herein, we demonstrate that DNA methyltransferases DNMT1 and DNMT3B are highly abundant in prostate cancer cells DU145, CWR22, LNCaP and PC-3; yet, these DNMTs bind primarily to the transcription suppressor in DU145, the only cells where methylation is critical for RAD9 regulation. For DU145 cells, DNMT1 or DNMT3B shRNA reduced RAD9 level and tumorigenicity, and RAD9 ectopic expression restored this latter activity in the DNMT knockdown cells. High levels of RAD9, DNMT1, DNMT3B and RAD9 transcription suppressor hypermethylation were significantly correlated in prostate tumors, and not in normal prostate tissues. Based on these results, we propose a novel model where RAD9 is regulated epigenetically by DNMT1 and DNMT3B, via targeted hypermethylation, and that consequent RAD9 overproduction promotes prostate tumorigenesis., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background  Recent outbreaks of duodenoscope-associated multidrug-resistant organisms (MDROs) have brought attention to the infection risk from procedures performed with duodenoscopes. Prior to these MDRO outbreaks, procedures with duodenoscopes were considered safe and low risk for exogenous infection transmission, provided they were performed in strict accordance with manufacturer instructions for use and multisociety reprocessing guidelines. The attention and efforts of the scientific community, regulatory agencies, and the device industry have deepened our understanding of factors responsible for suboptimal outcomes. These include instrument design, reprocessing practices, and surveillance strategies for detecting patient and instrument colonization. Various investigations have made it clear that current reprocessing methods fail to consistently deliver a pathogen-free instrument. The magnitude of infection transmission has been underreported due to several factors. These include the types of organisms responsible for infection, clinical signs presenting in sites distant from ERCP inoculation, and long latency from the time of acquisition to infection. Healthcare providers remain hampered by the ill-defined infectious risk innate to the current instrument design, contradictory information and guidance, and limited evidence-based interventions or reprocessing modifications that reduce risk. Therefore, the objectives of this narrative review included identifying outbreaks described in the peer-reviewed literature and comparing the findings with infections reported elsewhere. Search strategies included accessing peer-reviewed articles, governmental databases, abstracts for scientific conferences, and media reports describing outbreaks. This review summarizes current knowledge, highlights gaps in traditional sources of evidence, and explores opportunities to improve our understanding of actual risk and evidence-based approaches to mitigate risk., Competing Interests: Competing interests Ofstead, Buro, Hopkins, Eiland and Wetzler have received research grants, study materials, educational materials, or consulting contracts from 3 M Company, Ambu, Auris Health, Advanced Sterilization Products, Boston Scientific Corporation, Cogentix, Convergascent, Endoscopy Repair Specialists, Fortive, Healthmark, Invendo Medical, Laborie, Medivators, Mobile Instrument, Nanosonics, and Steris. Dr. Lichtenstein reports personal fees from Olympus America Inc. and Boston Scientific Corporation outside of the submitted work; has received consulting or speaking contracts from Augmenix, Iterative Scopes, Aries Pharmaceutical, and GI Supply; and has served as a principal investigator and advisor for Iterative Scopes and Motus., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commecial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

Background: Wound infections result in considerable morbidity, mortality and healthcare costs. Antibiotic resistance has complicated wound healing, and new, non-antibiotic-based treatment methods are being developed., Aims: To evaluate evidence on the safety, efficacy and real-world effectiveness of electroceutical devices (ECDs) that provide continuous electrical stimulation to wounds., Method: A systematic search was conducted to identify primary studies published between 2009 and 2019 that described therapeutic wound treatment using portable ECDs. Studies were included if the ECD delivered continuous electrical current directly to the wound area for the duration of treatment., Results: Of 171 citations identified in the search, 13 articles met the inclusion criteria and were analysed. Nine studies evaluated dressings embedded with zinc and silver particles that generated electricity electrochemically, and four evaluated electrode-based units with external batteries. ECDs were effective in healing complex, hard-to-heal wounds that had not responded to other treatments. Four studies showed that ECDs led to complete closure of wounds without complications, and in some cases healed wounds faster than standard of care (SOC). One study found that ECDs resulted in higher ratings by both patients and surgeons than SOC for the progression of wound healing and scar appearance. Additionally, three studies found ECD treatment was less expensive than SOC, due to patients requiring fewer dressing changes or nurse visits., Conclusion: ECDs appeared to be a safe, effective and cost-effective method for treating severe, complex and challenging wounds, including hard-to-heal wounds, surgical incisions and skin graft donor sites.

Endoscope reprocessing is often ineffective, and microbes frequently remain on endoscopes after the use of high-level disinfectants (HLDs). Several factors impact reprocessing effectiveness, including non-adherence to guidelines, use of damaged endoscopes, use of insoluble products during endoscopy, insufficient cleaning, contaminated rinse water, and inadequate drying before storage. Our team suspected that issues with HLD chemistries and monitoring could also contribute to reprocessing failures. We conducted a mixed-methods analysis of published literature, our interviews with frontline personnel, and evidence from our previous studies. The evidence showed that reusable HLDs commonly failed tests for minimum effective concentration (MEC) before their maximum usage periods. MEC tests also detected failures associated with single-use HLDs that did not fully deploy. These failures were due to product issues, process complexities, and personnel non-adherence with guidelines and manufacturer instructions. HLDs will likely continue to be used for the foreseeable future. More research is needed to assess real-world practice patterns related to the high-level disinfection step and MEC testing and to establish more realistic usage periods for reusable HLD chemistries. Manufacturers and researchers should evaluate the ability of technological solutions and engineered safeguards to overcome human error. Recognition of the need for quality improvement is growing, and infection preventionists should take action to build on this momentum and collaborate with manufacturers, endoscopists, and reprocessing personnel to improve the effectiveness of high-level disinfection., (Copyright © 2019 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.)

Flexible endoscopes are exposed to blood, mucus, and other secretions during procedures. Single-use sheaths are designed to prevent contact between contaminants and reusable endoscope components. This systematic review examined findings from 22 studies that assessed endoscopic sheath use during urologic, gastrointestinal, or respiratory procedures. The evidence showed that sheaths were durable and yielded faster endoscope turnover times because their reusable components did not require high-level disinfection or sterilization. After a brief learning period, health care providers successfully assembled and maneuvered sheathed endoscopes. Patients generally did not experience greater discomfort during procedures in which sheaths were used. Microbial cultures of sheathed endoscopes were negative or similar to unsheathed endoscopes. More research is needed to evaluate the potential effect of disposable sheaths on infection risks. The evidence suggests that sheaths are a viable option for reliably providing a barrier between endoscopes and patients without affecting the quality of endoscopic procedures., (© AORN, Inc, 2019.)

Background: Current methods for reprocessing flexible endoscopes do not consistently eliminate organic soil. The off-label use of simethicone as a defoaming agent may contribute to reprocessing failures, and endoscope manufacturers have cautioned against its use., Methods: We sought evidence of simethicone use by interviewing hospital personnel, conducting audits, inspecting endoscopes, and conducting tests., Results: Researchers examined 69 fully reprocessed endoscopes in 4 hospitals. Microbial cultures were positive for ≥50% of endoscopes. Researchers observed cloudy, shimmery fluid resembling simethicone inside channels and under a duodenoscope elevator mechanism. Crystallized white fragments were observed protruding from a gastroscope water jet outlet. Oily, sticky residue was found on endoscopes, and a 3-dimensional mass was found inside an endoscopic ultrasound endoscope. Hospital personnel reported the use of simethicone, cooking oil and silicone sprays, and tissue glue during endoscopy., Discussion: The off-label use of defoaming agents, lubricants, and tissue glue is common and many endoscopists consider these products essential. Our findings suggest these substances are not removed during reprocessing and may impact reprocessing effectiveness., Conclusions: Infection preventionists should determine whether these products are used in their institutions and evaluate methods for removing them. New policies may be needed to support procedural success and effective endoscope reprocessing., (Copyright © 2019 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.)

RAD9A plays an important role in prostate tumorigenesis and metastasis-related phenotypes. The protein classically functions as part of the RAD9A-HUS1-RAD1 complex but can also act independently. RAD9A can selectively transactivate multiple genes, including CDKN1A and NEIL1 by binding p53-consensus sequences in or near promoters. RAD9A is overexpressed in human prostate cancer specimens and cell lines; its expression correlates with tumor progression. Silencing RAD9A in prostate cancer cells impairs their ability to form tumors in vivo and migrate as well as grow anchorage independently in vitro. We demonstrate herein that RAD9A transcriptionally controls AGR2, a gene aberrantly overexpressed in patients with metastatic prostate cancer. Transient or stable knockdown of RAD9A in PC-3 cells caused downregulation of AGR2 protein abundance. Reduced AGR2 protein levels were due to lower abundance of AGR2 mRNA. The AGR2 genomic region upstream of the coding initiation site contains several p53 consensus sequences. RAD9A bound specifically to the 5'-untranslated region of AGR2 in PC-3 cells at a partial p53 consensus sequence at position +3136 downstream from the transcription start site, determined by chromatin immunoprecipitation, followed by PCR amplification. Binding of RAD9A to the p53 consensus sequence was sufficient to drive AGR2 gene transcription, shown by a luciferase reporter assay. In contrast, when the RAD9A-binding sequence on the AGR2 was mutated, no luciferase activity was detected. Knockdown of RAD9A in PC-3 cells impaired cell migration and anchorage-independent growth. However, ectopically expressed AGR2 in RAD9A-depleted PC-3 cells restored these phenotypes. Our results suggest RAD9A drives metastasis by controlling AGR2 abundance., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Prostate cancer is a complex disease, with multiple subtypes and clinical presentations. Much progress has been made in recent years to understand the underlying genetic basis that drives prostate cancer. Such mechanistic information is useful for development of novel therapeutic targets, to identify biomarkers for early detection or to distinguish between aggressive and indolent disease, and to predict treatment outcome. Multiple tests have become available in recent years to address these clinical needs for prostate cancer. We describe several of these assays, summarizing test details, performance characteristics, and acknowledging their limitations. There is a pressing unmet need for novel biomarkers that can demonstrate improvement in these areas. We introduce one such candidate biomarker, RAD9, describe its functions in the DNA damage response, and detail why it can potentially fill this void. RAD9 has multiple roles in prostate carcinogenesis, making it potentially useful as a clinical tool for men with prostate cancer. RAD9 was originally identified as a radioresistance gene, and subsequent investigations revealed several key functions in the response of cells to DNA damage, including involvement in cell cycle checkpoint control, at least five DNA repair pathways, and apoptosis. Further studies indicated aberrant overexpression in approximately 45% of prostate tumors, with a strong correlation between RAD9 abundance and cancer stage. A causal relationship between RAD9 and prostate cancer was first demonstrated using a mouse model, where tumorigenicity of human prostate cancer cells after subcutaneous injection into nude mice was diminished when RNA interference was used to reduce the normally high levels of the protein. In addition to activity needed for the initial development of tumors, cell culture studies indicated roles for RAD9 in promoting prostate cancer progression by controlling cell migration and invasion through regulation of ITGB1 protein levels, and anoikis resistance by modulating AKT activation. Furthermore, RAD9 enhances the resistance of human prostate cancer cells to radiation in part by regulating ITGB1 protein abundance. RAD9 binds androgen receptor and inhibits androgen-induced androgen receptor's activity as a transcription factor. Moreover, RAD9 also acts as a gene-specific transcription factor, through binding p53 consensus sequences at target gene promoters, and this likely contributes to its oncogenic activity. Given these diverse and extensive activities, RAD9 plays important roles in the initiation and progression of prostate cancer and can potentially serve as a valuable biomarker useful in the management of patients with this disease., Competing Interests: Conflicts of Interest: The authors have no conflicts of interest to declare

The way cells respond to DNA damage is important since inefficient repair or misrepair of lesions can have deleterious consequences, including mutation, genomic instability, neurodegenerative disorders, premature aging, cancer or death. Whether damage occurs spontaneously as a byproduct of normal metabolic processes, or after exposure to exogenous agents, cells muster a coordinated, complex DNA damage response (DDR) to mitigate potential harmful effects. A variety of activities are involved to promote cell survival, and include DNA repair, DNA damage tolerance, as well as transient cell cycle arrest to provide time for repair before entry into critical cell cycle phases, an event that could be lethal if traversal occurs while damage is present. When such damage is prolonged or not repairable, senescence, apoptosis or autophagy is induced. One major level of DDR regulation occurs via the orchestrated transcriptional control of select sets of genes encoding proteins that mediate the response. p53 is a transcription factor that transactivates specific DDR downstream genes through binding DNA consensus sequences usually in or near target gene promoter regions. The profile of p53-regulated genes activated at any given time varies, and is dependent upon type of DNA damage or stress experienced, exact composition of the consensus DNA binding sequence, presence of other DNA binding proteins, as well as cell context. RAD9 is another protein critical for the response of cells to DNA damage, and can also selectively regulate gene transcription. The limited studies addressing the role of RAD9 in transcription regulation indicate that the protein transactivates at least one of its target genes, p21/waf1/cip1, by binding to DNA sequences demonstrated to be a p53 response element. NEIL1 is also regulated by RAD9 through a similar DNA sequence, though not yet directly verified as a bonafide p53 response element. These findings suggest a novel pathway whereby p53 and RAD9 control the DDR through a shared mechanism involving an overlapping network of downstream target genes. Details and unresolved questions about how these proteins coordinate or compete to execute the DDR through transcriptional reprogramming, as well as biological implications, are discussed.

RAD9 participates in DNA damage-induced cell cycle checkpoints and DNA repair. As a member of the RAD9-HUS1-RAD1 (9-1-1) complex, it can sense DNA damage and recruit ATR to damage sites. RAD9 binding can enhance activities of members of different DNA repair pathways, including NEIL1 DNA glycosylase, which initiates base excision repair (BER) by removing damaged DNA bases. Moreover, RAD9 can act independently of 9-1-1 as a gene-specific transcription factor. Herein, we show that mouse Rad9(-/-) relative to Rad9(+/+) embryonic stem (ES) cells have reduced levels of Neil1 protein. Also, human prostate cancer cells, DU145 and PC-3, knocked down for RAD9 demonstrate reduced NEIL1 abundance relative to controls. We found that Rad9 is required for Neil1 protein stability in mouse ES cells, whereas it regulates NEIL1 transcription in the human cells. RAD9 depletion enhances sensitivity to UV, gamma rays and menadione, but ectopic expression of RAD9 or NEIL1 restores resistance. Glycosylase/apurinic lyase activity was reduced in Rad9(-/-) mouse ES and RAD9 knocked-down human prostate cancer whole cell extracts, relative to controls. Neil1 or Rad9 addition restored this incision activity. Thus, we demonstrate that RAD9 regulates BER by controlling NEIL1 protein levels, albeit by different mechanisms in human prostate cancer versus mouse ES cells., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Radiation induced bystander effects are an important component of the overall response of cells to irradiation and are associated with human health risks. The mechanism responsible includes intra-cellular and inter-cellular signaling by which the bystander response is propagated. However, details of the signaling mechanism are not well defined., Methods: We measured the bystander response of Mrad9+/+ and Mrad9-/- mouse embryonic stem cells, as well as human H1299 cells with inherent or RNA interference-mediated reduced RAD9 levels after exposure to 1 Gy α particles, by scoring chromosomal aberrations and micronuclei formation, respectively. In addition, we used microarray gene expression analyses to profile the transcriptome of directly irradiated and bystander H1299 cells., Results: We demonstrated that Mrad9 null enhances chromatid aberration frequency induced by radiation in bystander mouse embryonic stem cells. In addition, we found that H1299 cells with reduced RAD9 protein levels showed a higher frequency of radiation induced bystander micronuclei formation, compared with parental cells containing inherent levels of RAD9. The enhanced bystander response in human cells was associated with a unique transcriptomic profile. In unirradiated cells, RAD9 reduction broadly affected stress response pathways at the mRNA level; there was reduction in transcript levels corresponding to genes encoding multiple members of the UVA-MAPK and p38MAPK families, such as STAT1 and PARP1, suggesting that these signaling mechanisms may not function optimally when RAD9 is reduced. Using network analysis, we found that differential activation of the SP1 and NUPR1 transcriptional regulators was predicted in directly irradiated and bystander H1299 cells. Transcription factor prediction analysis also implied that HIF1α (Hypoxia induced factor 1 alpha) activation by protein stabilization in irradiated cells could be a negative predictor of the bystander response, suggesting that local hypoxic stress experienced by cells directly exposed to radiation may influence whether or not they will elicit a bystander response in neighboring cells.

In mitotic cells, RAD9A functions in repairing DNA double-strand breaks (DSBs) by homologous recombination and facilitates the process by cell cycle checkpoint control in response to DNA damage. DSBs occur naturally in the germline during meiosis but whether RAD9A participates in repairing such breaks is not known. In this study, we determined that RAD9A is indeed expressed in the male germ line with a peak of expression in late pachytene and diplotene stages, and the protein was found associated with the XY body. As complete loss of RAD9A is embryonic lethal, we constructed and characterized a mouse strain with Stra8-Cre driven germ cell-specific ablation of Rad9a beginning in undifferentiated spermatogonia in order to assess its role in spermatogenesis. Adult mutant male mice were infertile or sub-fertile due to massive loss of spermatogenic cells. The onset of this loss occurs during meiotic prophase, and there was an increase in the numbers of apoptotic spermatocytes as determined by TUNEL. Spermatocytes lacking RAD9A usually arrested in meiotic prophase, specifically in pachytene. The incidence of unrepaired DNA breaks increased, as detected by accumulation of γH2AX and DMC1 foci on the axes of autosomal chromosomes in pachytene spermatocytes. The DNA topoisomerase IIβ-binding protein 1 (TOPBP1) was still localized to the sex body, albeit with lower intensity, suggesting that RAD9A may be dispensable for sex body formation. We therefore show for the first time that RAD9A is essential for male fertility and for repair of DNA DSBs during meiotic prophase I.

RAD9 regulates multiple cellular processes that influence genomic integrity, and for at least some of its functions the protein acts as part of a heterotrimeric complex bound to HUS1 and RAD1 proteins. RAD9 participates in DNA repair, including base excision repair, homologous recombination repair and mismatch repair, multiple cell cycle phase checkpoints and apoptosis. In addition, functions including the transactivation of downstream target genes, immunoglobulin class switch recombination, as well as 3'-5' exonuclease activity have been reported. Aberrant RAD9 expression has been linked to breast, lung, thyroid, skin and prostate tumorigenesis, and a cause-effect relationship has been demonstrated for the latter two. Interestingly, human RAD9 overproduction correlates with prostate cancer whereas deletion of Mrad9, the corresponding mouse gene, in keratinocytes leads to skin cancer. These results reveal that RAD9 protein can function as an oncogene or tumor suppressor, and aberrantly high or low levels can have deleterious health consequences. It is not clear which of the many functions of RAD9 is critical for carcinogenesis, but several alternatives are considered herein and implications for the development of novel cancer therapies based on these findings are examined.

RAD9 participates in promoting resistance to DNA damage, cell cycle checkpoint control, DNA repair, apoptosis, embryogenesis, and regulation of transcription. A paralogue of RAD9 (named RAD9B) has been identified. To define the function of mouse Rad9b (Mrad9b), embryonic stem (ES) cells with a targeted gene deletion were constructed and used to generate Mrad9b mutant mice. Mrad9b(-/-) embryos are resorbed after E7.5 while some of the heterozygotes die between E12.5 and a few days after birth. Mrad9b is expressed in embryonic brain and Mrad9b(+/-) embryos exhibit abnormal neural tube closure. Mrad9b(-/-) mouse embryonic fibroblasts are not viable. Mrad9b(-/-) ES cells are more sensitive to gamma rays and mitomycin C than Mrad9b(+/+) controls, but show normal gamma-ray-induced G2/M checkpoint control. There is no evidence of spontaneous genomic instability in Mrad9b(-/-) cells. Our findings thus indicate that Mrad9b is essential for embryonic development and mediates resistance to certain DNA damaging agents., (© 2010 Wiley-Liss, Inc.)

Background: Cells are constantly exposed to stresses from cellular metabolites as well as environmental genotoxins. DNA damage caused by these genotoxins can be efficiently fixed by DNA repair in cooperation with cell cycle checkpoints. Unrepaired DNA lesions can lead to cell death, gene mutation and cancer. The Rad1 protein, evolutionarily conserved from yeast to humans, exists in cells as monomer as well as a component in the 9-1-1 protein complex. Rad1 plays crucial roles in DNA repair and cell cycle checkpoint control, but its contribution to carcinogenesis is unknown., Results: To address this question, we constructed mice with a deletion of Mrad1. Matings between heterozygous Mrad1 mutant mice produced Mrad1+/+ and Mrad1+/- but no Mrad1-/- progeny, suggesting the Mrad1 null is embryonic lethal. Mrad1+/- mice demonstrated no overt abnormalities up to one and half years of age. DMBA-TPA combinational treatment was used to induce tumors on mouse skin. Tumors were larger, more numerous, and appeared earlier on the skin of Mrad1+/- mice compared to Mrad1+/+ animals. Keratinocytes isolated from Mrad1+/- mice had significantly more spontaneous DNA double strand breaks, proliferated slower and had slightly enhanced spontaneous apoptosis than Mrad1+/+ control cells., Conclusion: These data suggest that Mrad1 is important for preventing tumor development, probably through maintaining genomic integrity. The effects of heterozygous deletion of Mrad1 on proliferation and apoptosis of keratinocytes is different from those resulted from Mrad9 heterozygous deletion (from our previous study), suggesting that Mrad1 also functions independent of Mrad9 besides its role in the Mrad9-Mrad1-Mhus1 complex in mouse cells.

The Rad9 gene is evolutionarily conserved from yeast to humans and plays crucial roles in genomic maintenance, DNA repair, and cell cycle checkpoint controls. However, the function of this gene with respect to tumorigenesis is not well-understood. A Rad9-null mutation in mice causes embryonic lethality. In this study, we created mice in which mouse Rad9, Mrad9, was deleted only in keratinocytes to permit examination of the potential function of the gene in tumor development. Mice with Mrad9(+/-) or Mrad9(-/-) keratinocytes showed no overt, spontaneous morphologic defects and seemed similar to wild-type controls. Painting the carcinogen 7,12-dimethylbenzanthracene (DMBA) onto the skin of the animals caused earlier onset and more frequent formation of tumors and senile skin plaques in Mrad9(-/-) mice, compared with Mrad9(+/-) and Mrad9(+/+) littermates. DNA damage response genes p21, p53, and Mrad9B were expressed at higher levels in Mrad9(-/-) relative to Mrad9(+/+) skin. Keratinocytes isolated from Mrad9(-/-) skin had more spontaneous and DMBA-induced DNA double strand breaks than Mrad9(+/+) keratinocytes, and the levels were reduced by incubation with the antioxidant epigallocatechin gallate. These data suggest that Mrad9 plays an important role in maintaining genomic stability and preventing tumor development in keratinocytes.

Rad9 and Atm regulate multiple cellular responses to DNA damage, including cell cycle checkpoints, DNA repair and apoptosis. However, the impact of dual heterozygosity for Atm and Rad9 is unknown. Using 50 cGy of X rays as an environmental insult and cataractogenesis as an end point, this study examined the effect of heterozygosity for one or both genes in mice. Posterior subcapsular cataracts, characteristic of radiation exposure, developed earlier in X-irradiated double heterozygotes than in single heterozygotes, which were more prone to cataractogenesis than wild-type controls. Cataract onset time and progression in single or double heterozygotes were accelerated even in unirradiated eyes. These findings indicate that the cataractogenic effect of combined heterozygosity is greater than for each gene alone and are the first to demonstrate the impact of multiple haploinsufficiency on radiation effects in an intact mammal. These observations may help explain observed interindividual differential radiosensitivity in human populations and have important implications for those undergoing radiotherapy or exposed to elevated levels of cosmic radiation, such as the astronaut corps. These findings demonstrate that Mrad9 and Atm are important determinants of lens opacification and, given the roles of Atm and Rad9 in maintaining genomic stability, are consistent with a genotoxic basis for radiation cataractogenesis.

The protein products of several rad checkpoint genes of Schizosaccharomyces pombe (rad1+, rad3+, rad9+, rad17+, rad26+, and hus1+) play crucial roles in sensing changes in DNA structure, and several function in the maintenance of telomeres. When the mammalian homologue of S. pombe Rad9 was inactivated, increases in chromosome end-to-end associations and frequency of telomere loss were observed. This telomere instability correlated with enhanced S- and G2-phase-specific cell killing, delayed kinetics of gamma-H2AX focus appearance and disappearance, and reduced chromosomal repair after ionizing radiation (IR) exposure, suggesting that Rad9 plays a role in cell cycle phase-specific DNA damage repair. Furthermore, mammalian Rad9 interacted with Rad51, and inactivation of mammalian Rad9 also resulted in decreased homologous recombinational (HR) repair, which occurs predominantly in the S and G2 phases of the cell cycle. Together, these findings provide evidence of roles for mammalian Rad9 in telomere stability and HR repair as a mechanism for promoting cell survival after IR exposure.

Two signaling pathways are activated by antineoplastic therapies that damage DNA and stall replication. In one pathway, double-strand breaks activate ataxia-telangiectasia mutated kinase (ATM) and checkpoint kinase 2 (Chk2), two protein kinases that regulate apoptosis, cell-cycle arrest, and DNA repair. In the second pathway, other types of DNA lesions and replication stress activate the Rad9-Hus1-Rad1 complex and the protein kinases ataxia-telangiectasia mutated and Rad3-related kinase (ATR) and checkpoint kinase 1 (Chk1), leading to changes that block cell-cycle progression, stabilize stalled replication forks, and influence DNA repair. Gemcitabine and cytarabine are two highly active chemotherapeutic agents that disrupt DNA replication. Here, we examine the roles these pathways play in tumor cell survival after treatment with these agents. Cells lacking Rad9, Chk1, or ATR were more sensitive to gemcitabine and cytarabine, consistent with the fact that these agents stall replication forks, and this sensitization was independent of p53 status. Interestingly, ATM depletion sensitized cells to gemcitabine and ionizing radiation but not cytarabine. Together, these results demonstrate that 1) gemcitabine triggers both checkpoint signaling pathways, 2) both pathways contribute to cell survival after gemcitabine-induced replication stress, and 3) although gemcitabine and cytarabine both stall replication forks, ATM plays differential roles in cell survival after treatment with these agents.

Loss of function of oncogenes, tumor suppressor genes and DNA damage processing genes has been implicated in the development of many types of cancer, but for the vast majority of cases, there is no link to specific germ line mutations. In the last several years, heterozygosity leading to haploinsufficiency for proteins involved in DNA repair pathways was shown to play a role in genomic instability and carcinogenesis after DNA damage is induced. Because the effect of haploinsufficiency for one protein is relatively small, we hypothesize that predisposition to cancer could be a result of the additive effect of heterozygosity for two or more genes, critical for pathways that control DNA damage signaling, repair or apoptosis. To address this issue, primary mouse cells, haploinsufficient for one or two proteins, ATM and RAD9, related to the cellular response to DNA damage were examined. The results show that cells having low levels of both ATM and RAD9 proteins are more sensitive to transformation by radiation, have different DNA double-strand break repair dynamics and are less apoptotic when compared with wild-type controls or those cells haploinsufficient for only one of these proteins. Our conclusions are that under stress conditions, the efficiency and capacity for DNA repair mediated by the ATM/RAD9 cell signaling network depend on the abundance of both proteins and that, in general, DNA repair network efficiencies are genotype-dependent and can vary within a specific range.

The fission yeast Schizosaccharomyces pombe rad9 gene promotes cell survival through activation of cell cycle checkpoints induced by DNA damage. Mouse embryonic stem cells with a targeted deletion of Mrad9, the mouse ortholog of this gene, were created to evaluate its function in mammals. Mrad9(-/-) cells demonstrated a marked increase in spontaneous chromosome aberrations and HPRT mutations, indicating a role in the maintenance of genomic integrity. These cells were also extremely sensitive to UV light, gamma rays, and hydroxyurea, and heterozygotes were somewhat sensitive to the last two agents relative to Mrad9(+/+) controls. Mrad9(-/-) cells could initiate but not maintain gamma-ray-induced G(2) delay and retained the ability to delay DNA synthesis rapidly after UV irradiation, suggesting that checkpoint abnormalities contribute little to the radiosensitivity observed. Ectopic expression of Mrad9 or human HRAD9 complemented Mrad9(-/-) cell defects, indicating that the gene has radioresponse and genomic maintenance functions that are evolutionarily conserved. Mrad9(+/-) mice were generated, but heterozygous intercrosses failed to yield Mrad9(-/-) pups, since embryos died at midgestation. Furthermore, Mrad9(-/-) mouse embryo fibroblasts were not viable. These investigations establish Mrad9 as a key mammalian genetic element of pathways that regulate the cellular response to DNA damage, maintenance of genomic integrity, and proper embryonic development.

When human cells incur DNA damage, two fundamental responses can follow, cell cycle arrest or apoptosis. Human RAD9 (hRAD9) and p53 function in both processes, but the mechanistic relationship between their activities is unknown. p53 mediates checkpoint control at G(1) by transcriptional regulation of p21. In this report, we show that hRAD9, like p53, can also regulate p21 at the transcriptional level. We demonstrate that overexpression of hRAD9 leads to increased p21 RNA and encoded protein levels. The promoter region of p21 fused to a luciferase reporter can be transactivated by either hRAD9 or p53, indicating that hRAD9 regulates the p21 promoter for transcriptional control of expression. Using an electrophoretic mobility-shift assay, we show that hRAD9 specifically binds to a p53-consensus DNA-binding sequence in the p21 promoter. Microarray screening coupled with Northern analysis reveals that hRAD9 regulates the abundance of other messages in addition to p21. Our data reveal a previously undescribed mechanism for regulation of p21 and demonstrate that hRAD9 can control gene transcription. We suggest that hRAD9 and p53 co-regulate p21 to direct cell cycle progression by similar molecular mechanisms. Furthermore, hRAD9 might regulate other cellular processes as well by modulating transcription of multiple down-stream target genes.

Previous studies have suggested two possible roles for Rad9 in mammalian cells subjected to replication stress or DNA damage. One model suggests that a Rad9-containing clamp is loaded onto damaged DNA, where it participates in Chk1 activation and subsequent events that contribute to cell survival. The other model suggests that Rad9 translocates to mitochondria, where it triggers apoptosis by binding to and inhibiting Bcl-2 and Bcl-x(L). To further study the role of Rad9, parental and Rad9(-/-) murine embryonic stem (ES) cells were treated with camptothecin, etoposide, or cytarabine, all prototypic examples of three classes of widely used anticancer agents. All three agents induced Rad9 chromatin binding. Each of these agents also triggered S-phase checkpoint activation in parental ES cells, as indicated by a caffeine-inhibitable decrease in [3H]thymidine incorporation into DNA and Cdc25A down-regulation. Interestingly, the ability of cytarabine to activate the S-phase checkpoint was severely compromised in Rad9(-/-) cells, whereas activation of this checkpoint by camptothecin and etoposide was unaltered, suggesting that the action of cytarabine is readily distinguished from that of classical topoisomerase poisons. Nonetheless, Rad9 deletion sensitized ES cells to the cytotoxic effects of all three agents, as evidenced by enhanced apoptosis and diminished colony formation. Collectively, these results suggest that the predominant role of Rad9 in ES cells is to promote survival after replicative stress and topoisomerase-mediated DNA damage.

Human and mouse paralogues of the evolutionarily conserved mammalian HRAD9 and Mrad9 cell cycle checkpoint control genes have been isolated and called HRAD9B and Mrad9B, respectively. HRAD9B encodes a protein that is 414 amino acids long and is 55% similar and 35% identical to the HRAD9 gene product. The Mrad9B protein is 398 amino acids long and is 50% similar and 35% identical to its paralogue. We demonstrate that the encoded human protein is nuclear and can physically interact with checkpoint proteins HRAD1, HRAD9, HHUS1, and HHUS1B, much like HRAD9. Northern blot analysis to detect tissue specificity indicates that the human and mouse genes are expressed predominantly in the testis. The abundance of HRAD9B RNA, as judged by quantitative reverse transcription-PCR, is very low in most testicular tumors, particularly those of germ cell origin, i.e., seminomas, relative to normal testis control, nonseminomas, or Leydig tumor cells. RNA levels corresponding to HRAD17, another checkpoint control gene, demonstrated a similar pattern, but in general, higher quantities of this message were detected in samples. Furthermore, normal/tumor tissue differences were not as dramatic or consistent from sample to sample, especially for the seminomas. Our results demonstrate for the first time that HRAD9 and Mrad9 are part of a gene family and reveal a new genetic element encoding a product that interacts with multiple, known cell cycle checkpoint control proteins. The findings also indicate that HRAD9B can serve as a biomarker in particular for testicular seminomas and might be causally related to the disease.

Rad9, a key component of genotoxin-activated checkpoint signaling pathways, associates with Hus1 and Rad1 in a heterotrimeric complex (the 9-1-1 complex). Rad9 is inducibly and constitutively phosphorylated. However, the role of Rad9 phosphorylation is unknown. Here we identified nine phosphorylation sites, all of which lie in the carboxyl-terminal 119-amino acid Rad9 tail and examined the role of phosphorylation in genotoxin-triggered checkpoint activation. Rad9 mutants lacking a Ser-272 phosphorylation site, which is phosphorylated in response to genotoxins, had no effect on survival or checkpoint activation in Mrad9-/- mouse ES cells treated with hydroxyurea (HU), ionizing radiation (IR), or ultraviolet radiation (UV). In contrast, additional Rad9 tail phosphorylation sites were essential for Chk1 activation following HU, IR, and UV treatment. Consistent with a role for Chk1 in S-phase arrest, HU- and UV-induced S-phase arrest was abrogated in the Rad9 phosphorylation mutants. In contrast, however, Rad9 did not play a role in IR-induced S-phase arrest. Clonogenic assays revealed that cells expressing a Rad9 mutant lacking phosphorylation sites were as sensitive as Rad9-/- cells to UV and HU. Although Rad9 contributed to survival of IR-treated cells, the identified phosphorylation sites only minimally contributed to survival following IR treatment. Collectively, these results demonstrate that the Rad9 phospho-tail is a key participant in the Chk1 activation pathway and point to additional roles for Rad9 in cellular responses to IR.

It has long been accepted that the radiation-induced heritable effects in mammalian cells are the result of direct DNA damage. Recent evidence, however, suggests that when a cell population is exposed to a low dose of alpha particles, biological effects occur in a larger proportion of cells than are estimated to have been traversed by alpha particles. Experiments involving the Columbia University microbeam, which allows a known fraction of cells to be traversed by a defined number of alpha particles, have demonstrated a bystander effect for clonogenic survival and oncogenic transformation in C3H 10T(1/2) cells. When 1 to 16 alpha particles were passed through the nuclei of 10% of a C3H 10T(1/2) cell population, more cells were unable to form colonies than were actually traversed by alpha particles. Both hit and non-hit cells contributed to the outcome of the experiments. The present work was undertaken to assess the bystander effect of radiation in only non-hit cells. For this purpose, Chinese hamster V79 cells transfected with hygromycin- or neomycin-resistance genes were used. V79 cells stably transfected with a hygromycin resistance gene and stained with a nuclear dye were irradiated with the charged-particle microbeam in the presence of neomycin-resistant cells. The biological effect was studied in the neomycin-resistant V79 cells after selective removal of the hit cells with geneticin treatment.

A functional homolog (rhp23) of human HHR23A and Saccharomyces cerevisiae RAD23 was cloned from the fission yeast Schizosaccharomyces pombe and characterized. Consistent with the role of Rad23 homologs in nucleotide excision repair, rhp23 mutant cells are moderately sensitive to UV light but demonstrate wild-type resistance to gamma-rays and hydroxyurea. Expression of the rhp23, RAD23 or HHR23A cDNA restores UV resistance to the mutant, indicating that rhp23 is a functional homolog of the human and S.cerevisiae genes. The rhp23::ura4 mutation also causes a delay in the G2 phase of the cell cycle which is corrected when rhp23, RAD23 or HHR23A cDNA is expressed. Rhp23 is present throughout the cell but is located predominantly in the nucleus, and the nuclear levels of Rhp23 decrease around the time of S phase in the cell cycle. Rhp23 is ubiquitinated at low levels, but overexpression of the rhp23 cDNA induces a large increase in ubiquitination of other proteins. Consistent with a role in protein ubiquitination, Rhp23 binds ubiquitin, as determined by two-hybrid analysis. Thus, the rhp23 gene plays a role not only in nucleotide excision repair but also in cell cycle regulation and the ubiquitination pathways.

Schizosaccharomyces pombe rad9 mutations can render cells sensitive to hydroxyurea (HU), gamma-rays and UV light and eliminate associated checkpoint controls. In vitro mutagenesis was performed on S.pombe rad9 and altered alleles were transplaced into the genome to ascertain the functional significance of five groups of evolutionarily conserved amino acids. Most targeted regions were changed to alanines, whereas rad9-S3 encodes a protein devoid of 22 amino acids normally present in yeast but absent from mammalian Rad9 proteins. We examined whether these rad9 alleles confer radiation and HU sensitivity and whether the sensitivities correlate with checkpoint control deficiencies. One rad9 mutant allele was fully active, whereas four others demonstrated partial loss of function. rad9-S1, which contains alterations in a BH3-like domain, conferred HU resistance but increased sensitivity to gamma-rays and UV light, without affecting checkpoint controls. rad9-S2 reduced gamma-ray sensitivity marginally, without altering other phenotypes. Two alleles, rad9-S4 and rad9-S5, reduced HU sensitivity, radiosensitivity and caused aberrant checkpoint function. HU-induced checkpoint control could not be uncoupled from drug resistance. These results establish unique as well as overlapping functional domains within Rad9p and provide evidence that requirements of the protein for promoting resistance to radiation and HU are not identical.

Here we report that the Schizosaccharomyces pombe Rad9 (SpRad9) protein contains a group of amino acids with similarity to the Bcl-2 homology 3 death domain, which is required for SpRad9 interaction with human Bcl-2 and apoptosis induction in human cells. Overexpression of Bcl-2 in S. pombe inhibits cell growth independently of rad9, but enhances resistance of rad9-null cells to methyl methanesulfonate, ultraviolet and ionizing radiation. These observations suggest that SpRad9 may represent the first member of the Bcl-2 protein family identified in yeast, though the cell death pathways in S. pombe may differ from those found in mammals.

DNA damage induces apoptosis through a signalling pathway that can be suppressed by the BCL-2 protein, but the mechanism by which DNA damage does this is unknown. Here, using yeast two-hybrid and co-immunoprecipitation studies, we show that RAD9, a human protein involved in the control of a cell-cycle checkpoint, interacts with the anti-apoptotic Bcl-2-family proteins BCL-2 and BCL-x L, but not with the pro-apoptotic BAX and BAD. When overexpressed in mammalian cells, RAD9 induces apoptosis that can be blocked by BCL-2 or BCL-x L. Conversely, antisense RAD9 RNA suppresses cell death induced by methyl methanesulphonate. These findings indicate that RAD9 may have a new role in regulating apoptosis after DNA damage, in addition to its previously described checkpoint-control and other radioresistance-promoting functions.

We have isolated a murine cDNA, Mrad9, that is orthologous to the fission yeast rad9+ and human HRAD9 genes. Mrad9 encodes a 389 amino acid long, 42,032 Dalton protein that is 27% identical and 56% similar to Rad9p, and 82% identical and 88% similar to HRAD9, at the amino acid level. Expression of the Mrad9 cDNA in Schizosaccharomyces pombe rad9::ura4+ cells restores nearly wild-type levels of hydroxyurea resistance and early S phase checkpoint control to mutant fission yeast cell populations. However, UV resistance is only minimally restored, and mutant cells remain sensitive to gamma radiation. Mrad9 genomic DNA was isolated from a mouse 129/SvEv library. The Mrad9 gene was local ized to a 15-kbp genomic DNA fragment, and contains 10 exons separated by 9 introns. Northern blot analysis indicates that the gene is expressed in many different tissues of the adult mouse, but the mRNA is most abundant in the heart and present at very low levels in the liver. These studies demonstrate the existence of a murine orthologue of the fission yeast rad9+ gene and underscore at least the partial evolutionary conservation of rad9+-dependent checkpoint control mechanisms.

The human BLM gene is a member of the Escherichia coli recQ helicase family, which includes the Saccharomyces cerevisiae SGS1 and human WRN genes. Defects in BLM are responsible for the human disease Bloom's syndrome, which is characterized in part by genomic instability and a high incidence of cancer. Here we describe the cloning of rad12+, which is the fission yeast homolog of BLM and is identical to the recently reported rhq1+ gene. We showed that rad12 null cells are sensitive to DNA damage induced by UV light and gamma radiation, as well as to the DNA synthesis inhibitor hydroxyurea. Overexpression of the wild-type rad12+ gene also leads to sensitivity to these agents and to defects associated with the loss of the S-phase and G2-phase checkpoint control. We showed genetically and biochemically that rad12+ acts upstream from rad9+, one of the fission yeast G2 checkpoint control genes, in regulating exit from the S-phase checkpoint. The physical chromosome segregation defects seen in rad12 null cells combined with the checkpoint regulation defect seen in the rad12+ overproducer implicate rad12+ as a key coupler of chromosomal integrity with cell cycle progression.

The product of the Schizosaccharomyces pombe rad9+ gene is required for cell cycle arrest at the G2 checkpoints in response to incompletely replicated or damaged DNA. We have identified a human cDNA from an infant brain library that is a structural homolog of S. pombe rad9+, by searching the dBest data base for sequences similar to the fission yeast gene. The human gene encodes a 391-amino acid long, 42,520-Da protein that is approximately 25% identical and 52% similar to the yeast protein. The human and yeast gene products demonstrate partial conservation of function, as the human cDNA can rescue to different degrees the sensitivity of S. pombe rad9::ura4+ cells to the DNA synthesis inhibitor hydroxyurea and gamma rays, as well as the associated checkpoint controls. These results suggest an underlying conservation of the molecular mechanisms of S and G2 checkpoint control pathways in most if not all eukaryotes. Fluorescence in situ hybridization using a fragment of the corresponding human genome as a probe, in conjunction with PCR reactions employing DNA from human X rodent somatic cell hybrids, has localized the gene to human chromosome 11q13.1-13.2. This region contains a number of tumor suppressor loci, and based on the biology of checkpoint control genes, HRAD9 should be considered a strong candidate for one of them.