The impact of post-operative enteral nutrition on duodenal injury outcomes: A post hoc analysis of an EAST multicenter trial.

BACKGROUND: Leak following surgical repair of traumatic duodenal injuries results in prolonged hospitalization and oftentimes nil per os(NPO) treatment. Parenteral nutrition(PN) has known morbidity; however, duodenal leak(DL) patients often have complex injuries and hospital courses resulting in barriers to enteral nutrition(EN). We hypothesized EN alone would be associated with 1)shorter duration until leak closure and 2)less infectious complications and shorter hospital length of stay(HLOS) compared to PN. METHODS: This was a post-hoc analysis of a retrospective, multicenter study from 35 Level-1 trauma centers, including patients >14 years-old who underwent surgery for duodenal injuries(1/2010-12/2020) and endured post-operative DL. The study compared nutrition strategies: EN vs PN vs EN + PN using Chi-Square and Kruskal-Wallis tests; if significance was found pairwise comparison or Dunn's test were performed. RESULTS: There were 113 patients with DL: 43 EN, 22 PN, and 48 EN + PN. Patients were young(median age 28 years-old) males(83.2%) with penetrating injuries(81.4%). There was no difference in injury severity or critical illness among the groups, however there were more pancreatic injuries among PN groups. EN patients had less days NPO compared to both PN groups(12 days[IQR23] vs 40[54] vs 33[32],p = <0.001). Time until leak closure was less in EN patients when comparing the three groups(7 days[IQR14.5] vs 15[20.5] vs 25.5[55.8],p = 0.008). EN patients had less intra-abdominal abscesses, bacteremia, and days with drains than the PN groups(all p < 0.05). HLOS was shorter among EN patients vs both PN groups(27 days[24] vs 44[62] vs 45[31],p = 0.001). When controlling for predictors of leak, regression analysis demonstrated EN was associated with shorter HLOS(ß -24.9, 95%CI -39.0 to -10.7,p < 0.001). CONCLUSION: EN was associated with a shorter duration until leak closure, less infectious complications, and shorter length of stay. Contrary to some conventional thought, PN was not associated with decreased time until leak closure. We therefore suggest EN should be the preferred choice of nutrition in patients with duodenal leaks whenever feasible. LEVEL OF EVIDENCE: IV.

Outcomes among trauma patients with duodenal leak following primary versus complex repair of duodenal injuries: An Eastern Association for the Surgery of Trauma multicenter trial.

BACKGROUND: Duodenal leak is a feared complication of repair, and innovative complex repairs with adjunctive measures (CRAM) were developed to decrease both leak occurrence and severity when leaks occur. Data on the association of CRAM and duodenal leak are sparse, and its impact on duodenal leak outcomes is nonexistent. We hypothesized that primary repair alone (PRA) would be associated with decreased duodenal leak rates; however, CRAM would be associated with improved recovery and outcomes when leaks do occur. METHODS: A retrospective, multicenter analysis from 35 Level 1 trauma centers included patients older than 14 years with operative, traumatic duodenal injuries (January 2010 to December 2020). The study sample compared duodenal operative repair strategy: PRA versus CRAM (any repair plus pyloric exclusion, gastrojejunostomy, triple tube drainage, duodenectomy). RESULTS: The sample (N = 861) was primarily young (33 years) men (84%) with penetrating injuries (77%); 523 underwent PRA and 338 underwent CRAM. Complex repairs with adjunctive measures were more critically injured than PRA and had higher leak rates (CRAM 21% vs. PRA 8%, p < 0.001). Adverse outcomes were more common after CRAM with more interventional radiology drains, prolonged nothing by mouth and length of stay, greater mortality, and more readmissions than PRA (all p < 0.05). Importantly, CRAM had no positive impact on leak recovery; there was no difference in number of operations, drain duration, nothing by mouth duration, need for interventional radiology drainage, hospital length of stay, or mortality between PRA leak versus CRAM leak patients (all p > 0.05). Furthermore, CRAM leaks had longer antibiotic duration, more gastrointestinal complications, and longer duration until leak resolution (all p < 0.05). Primary repair alone was associated with 60% lower odds of leak, whereas injury grades II to IV, damage control, and body mass index had higher odds of leak (all p < 0.05). There were no leaks among patients with grades IV and V injuries repaired by PRA. CONCLUSION: Complex repairs with adjunctive measures did not prevent duodenal leaks and, moreover, did not reduce adverse sequelae when leaks did occur. Our results suggest that CRAM is not a protective operative duodenal repair strategy, and PRA should be pursued for all injury grades when feasible. LEVEL OF EVIDENCE: Therapeutic/Care Management; Level IV.

FOXP1 and NDRG1 act differentially as downstream effectors of RAD9-mediated prostate cancer cell functions.

Metastatic progression is the key feature of prostate cancer primarily responsible for mortality caused by this disease. RAD9 is an oncogene for prostate cancer, and the encoded protein enhances metastasis-related phenotypes. RAD9 is a transcription factor with a limited set of regulated target genes, but the complete list of downstream genes critical for prostate carcinogenesis is unknown. We used microarray gene expression profiling and chromatin immunoprecipitation in parallel to identify genes transcriptionally controlled by RAD9 that contribute to this cancer. We found expression of 44 genes altered in human prostate cancer DU145 cells when RAD9 is knocked down by siRNA, and all of them bind RAD9 at their genomic location. FOXP1 and NDRG1 were down regulated when RAD9 expression was reduced, and we evaluated them further. We demonstrate that reduced RAD9, FOXP1 or NDGR1 expression decreases cell proliferation, rapid migration, anchorage-independent growth, anoikis resistance, and aerobic glycolysis. Ectopic expression of FOXP1 or NDRG1 partially restored aerobic glycolysis to prostate cancer cells with reduced RAD9 abundance, but only FOXP1 significantly complemented the other deficiencies. We thus show, for the first time, that RAD9 regulates FOXP1 and NDRG1 expression, and they function differently as downstream effectors for RAD9-mediated prostate cancer cell activities.

DNMT1 and DNMT3B regulate tumorigenicity of human prostate cancer cells by controlling RAD9 expression through targeted methylation.

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.

Colonic perforation due to inadvertent intraperitoneal LVAD driveline placement.

BACKGROUND: Left ventricular assist devices (LVAD) are placed for patients with advanced heart failure or cardiogenic shock as destination therapy or as a bridge to cardiac transplantation. Significant complications associated with LVAD placement include bleeding, infection, pump thrombosis, right heart failure, device malfunction and stroke. The case below illustrates inadvertent intraperitoneal driveline placement causing colonic perforation and the subsequent management. CASE PRESENTATION: A 54 year old male with a history of Wolff-Parkinson-White syndrome resulting in multiple readmissions for heart failure, ultimately required placement of a left ventricular assist device (LVAD). Several weeks later, he was found to have stool draining from the driveline site. The patient was taken to the operating room for limited exploration by the Cardiothoracic Surgery team and a bowel injury was identified and repaired. Three days after this repair, stool was once again leaking from the driveline site, requiring re-exploration by the Acute Care Surgery team. Intraoperatively, the prior repair was found to be leaking and multiple intra-abdominal abscesses were discovered. The transverse colon was resected and left in discontinuity. On a planned second look operation, the LVAD driveline was relocated to be extra-peritoneal and a colostomy was formed. DISCUSSION AND CONCLUSION: This case demonstrates the importance of early recognition and involvement of an Acute Care Surgeon in the management of this complex problem. Appropriate treatment involves a complete exploration, source control, driveline relocation and possible fecal diversion. Although the incidence of this complication is low, it must be considered in the differential in a septic LVAD patient.

Targeting MEK5 impairs nonhomologous end-joining repair and sensitizes prostate cancer to DNA damaging agents.

Radiotherapy is commonly used to treat a variety of solid human tumors, including localized prostate cancer. However, treatment failure often ensues due to tumor intrinsic or acquired radioresistance. Here we find that the MEK5/ERK5 signaling pathway is associated with resistance to genotoxic stress in aggressive prostate cancer cells. MEK5 knockdown by RNA interference sensitizes prostate cancer cells to ionizing radiation (IR) and etoposide treatment, as assessed by clonogenic survival and short-term proliferation assays. Mechanistically, MEK5 downregulation impairs phosphorylation of the catalytic subunit of DNA-PK at serine 2056 in response to IR or etoposide treatment. Although MEK5 knockdown does not influence the initial appearance of radiation- and etoposide-induced γH2AX and 53BP1 foci, it markedly delays their resolution, indicating a DNA repair defect. A cell-based assay shows that nonhomologous end joining (NHEJ) is compromised in cells with ablated MEK5 protein expression. Finally, MEK5 silencing combined with focal irradiation causes strong inhibition of tumor growth in mouse xenografts, compared with MEK5 depletion or radiation alone. These findings reveal a convergence between MEK5 signaling and DNA repair by NHEJ in conferring resistance to genotoxic stress in advanced prostate cancer and suggest targeting MEK5 as an effective therapeutic intervention in the management of this disease.

Ballistic and Penetrating Injuries of the Chest.

Ballistic injuries are a major cause of morbidity and mortality in the United States. Unstable patients have high mortality, and only a small subset arrive at the hospital alive. Many patients undergo emergent surgery upon arrival, but a small subset undergo imaging with plain film, computed tomography, and echocardiography. We present a pictorial essay of ballistic and penetrating injuries and their complications with a focus on lung, cardiac, and vascular injury.

Pexy of intraperitoneal LVAD driveline to relieve small bowel obstruction.

BACKGROUND: Left ventricular assist device (LVAD) implantation is a lifesaving intervention in advanced heart failure. However, LVAD is not without complication. In this case, an inadvertent intraperitoneal driveline caused small bowel obstruction, subsequently requiring pexy of the driveline to the abdominal wall to avoid future complications. CASE PRESENTATION: A 37-year-old male with worsening, nonischemic, dilated cardiomyopathy underwent LVAD implantation. Postoperative day (POD) 15 he developed small bowel obstruction, and abdominal exploration showed transition point at an inadvertently placed intraperitoneal LVAD driveline. The patient was LVAD-dependent precluding removal, so the driveline was secured to the anterior abdominal wall. He subsequently improved and was discharged. CONCLUSIONS: While LVAD is increasingly common for heart failure patients, the tunneled driveline may inadvertently enter the peritoneal cavity where it can cause significant morbidity. In this case, we propose securing the driveline to the abdominal wall to prevent complications when LVAD removal is not an option.

A survey of the practice and attitudes of surgeons regarding the treatment of appendicitis.

BACKGROUND: We surveyed surgeons to document their attitudes, practice, and risk tolerance regarding the treatment of appendicitis. METHODS: A web-based survey was sent to the EAST membership. The primary composite endpoint was defined as 1-year incidence of perioperative complications, antibiotic failure, infections, ED visits, and readmissions. RESULTS: A total of 563 of 1645 surveys were completed (34% response). Mean age was 47 ±â€¯10 years and 98% were from the United States. Most (72%) were employed at academic teaching hospitals and 66% practiced in an urban setting. There were significant differences in treatment recommendations for different presentations of appendicitis. Regarding the primary composite endpoint, surgeons would tolerate a median 17% [10%-25%] excess morbidity in order to avoid an operation (i.e. non-inferiority) and would require a median 24% [10%-50% lower morbidity for the surgical approach in order to declare it a superior treatment (i.e. superiority). CONCLUSIONS: To be considered non-inferior, antibiotic therapy of appendicitis cannot have >17% excess morbidity and appendectomy must have at least 24% lower morbidity to be considered superior.

RAD9A promotes metastatic phenotypes through transcriptional regulation of anterior gradient 2 (AGR2).

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.

Prostate cancer: unmet clinical needs and RAD9 as a candidate biomarker for patient management.

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.

Bowel obstruction due to retained intraperitoneal left ventricular assist device (LVAD) driveline.

BACKGROUND: Left ventricular assist devices (LVAD) provide a lifesaving bridge to cardiac transplant. Utilization of these devices is increasing in the United States. When a patient undergoes cardiac transplant, the left ventricular device is surgically removed and the driveline is extracted or left tunneled in the subcutaneous tissue. Our group encountered a rare and previously unreported complication of this device: intraperitoneal infiltration of a retained driveline after cardiac transplant causing a small bowel obstruction. CASE PRESENTATION: A 62 year old male with a past medical history of non-ischemic cardiomyopathy induced heart failure, status post bridging left ventricular assist device and orthotopic heart transplant presented with abdominal distention, tenderness, and leukocytosis six days post-transplant. CT abdomen and pelvis revealed dilated loops of bowel, air-fluid levels and a transition point in the proximal small bowel. The patient was diagnosed with small bowel obstruction and taken for exploratory laparotomy. He was found to have a retained intraabdominal LVAD driveline strangulating a loop of small bowel in the left upper quadrant. The driveline was removed and the section of bowel released with return of perfusion. CONCLUSIONS: We had encountered a rare complication of retained left ventricular assist device driveline after cardiac transplant: inadvertent penetration into the peritoneal cavity resulting in strangulation of small bowel. This complication, though uncommon, provides substantial risk to patients previously treated with left ventricular assist devices. Meticulous care must be taken to ensure proper device insertion and extraction, as well as consideration of this etiology when patients present with bowel obstruction after cardiac transplant.

Same-Hospital Re-Admission Rate Is Not Reliable for Measuring Post-Operative Infection-Related Re-Admission.

BACKGROUND: Post-operative infections cause morbidity, consume resources, and are an important quality measure in assessing and comparing hospitals. Commonly used metrics do not account for re-admission to a different hospital. The Nationwide Readmissions Database (NRD) tracks re-admissions across United States (US) hospitals. Infection-related re-admission across US hospitals has not been studied previously. PATIENTS AND METHODS: The 2013 NRD was queried for admissions with a primary International Classification of Diseases and Related Health Problems, 9th revision, Clinical Modification code for the most frequently performed operations. Non-elective all-cause, infection-related, and different hospital 30-day re-admission rates were calculated, using All Patient Refined Diagnosis Related Groups codes. Multi-variable logistic regression identified risk factors for re-admission. RESULTS: Of 826,836 surviving to discharge, 39,281 (4.8%) had an unplanned re-admission within 30 days, occurring at a different hospital 20.5% of the time. The most common reason for re-admission was infection (25.1%). Orthopedic and spinal procedures were at highest risk for all-cause and infection-related different hospital re-admission. Infection-related different hospital re-admission risk factors included: Length of stay >30 days (odds ratio [OR] 2.28 [1.62-3.21], p < 0.01), age ≥65 years (OR 1.56 [1.38-1.76], p < 0.01), and Charlson Comorbidity Index >1 (OR 1.14 [1.01-1.28], p < 0.01) and differed from predictors of same-hospital infectious re-admission. Non-elective surgical procedure (OR 0.79 [0.72-0.87], p < 0.01) and initial hospitalization at a large hospital (OR 0.66 [0.59-0.74], p < 0.01) were protective. CONCLUSION: A substantial proportion of post-operative re-admissions are missed by same-hospital re-admission data. All-cause and infection-related post-operative re-admissions to a different hospital are affected by unique patient and institution-specific factors. Re-admission reduction programs, quality metrics, and policy based on same hospital re-admission data should be updated to incorporate different hospital re-admission.

p53 and RAD9, the DNA Damage Response, and Regulation of Transcription Networks.

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.

Anti-Xa-guided enoxaparin thromboprophylaxis reduces rate of deep venous thromboembolism in high-risk trauma patients.

BACKGROUND: Appropriate prophylaxis against venous thromboembolism (VTE) remains undefined. This study evaluated an anti-Xa-guided enoxaparin thromboprophylaxis (TPX) protocol on the incidence of VTE in high-risk trauma patients based on Greenfield's Risk Assessment Profile (RAP) score. METHODS: This is a retrospective observational study of patients admitted to a trauma intensive care unit over a 12-month period. Patients were included if they received anti-Xa-guided enoxaparin TPX. Dosage was adjusted to a prophylactic peak anti-Xa level of 0.2 to 0.4 IU/mL. Subgroup analysis was performed on high-risk patients (RAP score ≥10) who received lower-extremity duplex ultrasound surveillance for deep vein thrombosis (DVT). Data are expressed as mean ± SD. Significance was assessed at p < 0.05. RESULTS: One hundred thirty-one patients received anti-Xa-guided enoxaparin TPX. Four patients were excluded for age or acute VTE on admission. Fifty-six patients with RAP score of ≥10 and surveillance duplex evaluations were included in the subgroup analysis with mean age 43 ± 20 years, Injury Severity Score of 25 ± 10, and RAP score of 16 ± 4. Prophylactic anti-Xa levels were initially achieved in 34.6% of patients. An additional 25.2% required 40 to 60 mg twice daily to reach prophylactic levels; 39.4% never reached prophylactic levels. Weight, body mass index, ISS, and RAP score were significantly higher with subprophylactic anti-Xa levels. One patient developed bleeding complications (0.8%). No patient developed intracerebral bleeding or heparin-induced thrombocytopenia.Nine VTE events occurred in the high-risk subgroup, including four DVT (7.1%), all asymptomatic, and five pulmonary emboli (8.9%). The historical rate of DVT in similar patients (ISS 31 ± 12 and RAP score 16 ± 5) was 20.5%, a significant decrease (p = 0.031). Mean chest Abbreviated Injury Scale scores were significantly higher for patients developing pulmonary emboli than DVT, 3.0 ± 1.1 vs. 0.0 (p < 0.001). CONCLUSIONS: Mean chest Abbreviated Injury Scale score was higher in patients developing pulmonary embolism. Increased weight, body mass index, ISS, and RAP score are associated with subprophylactic anti-Xa levels. Anti-Xa-guided enoxaparin dosing reduced the rate of DVT from 20.5% to 7.1% in high-risk trauma patients. LEVEL OF EVIDENCE: Therapeutic study, level IV.

Regulation of NEIL1 protein abundance by RAD9 is important for efficient base excision repair.

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.

RAD9 deficiency enhances radiation induced bystander DNA damage and transcriptomal response.

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.

RAD9 enhances radioresistance of human prostate cancer cells through regulation of ITGB1 protein levels.

BACKGROUND: Mouse embryonic stem cells null for Rad9 are sensitive to deleterious effects of ionizing radiation exposure. Likewise, integrin ß1 is a known radioprotective factor. Previously, we showed that RAD9 downregulation in human prostate cancer cells reduces integrin ß1 protein levels and ectopic expression of Mrad9 restores inherent high levels. METHODS: We used RNA interference to knockdown Rad9 expression in PC3 and DU145 prostate cancer cells. These cells were then exposed to ionizing radiation, and integrin ß1 protein levels were measured by immunoblotting. Survival of irradiated cells was measured by clonogenicity, cell cycle analysis, PARP-1 cleavage, and trypan blue exclusion. RESULTS: The function of RAD9 in controlling integrin ß1 expression is unique and not shared by the other members of the 9-1-1 complex, HUS1 and RAD1. RAD9 or integrin ß1 silencing sensitizes DU145 and PC3 cells to ionizing radiation. Irradiation of DU145 cells with low levels of RAD9 induces cleavage of PARP-1 protein. High levels of ionizing radiation have no effect on integrin ß1 protein levels. However, when RAD9 downregulation is combined with 10 Gy of ionizing radiation in DU145 or PC3 cells, there is an additional 50% downregulation of integrin ß1 compared with levels in unirradiated RAD9 knockdown cells. Finally, PC3 cells growing on fibronectin display increased radioresistance. However, PC3 cells with RAD9 knockdown are no longer protected by fibronectin after treatment with ionizing radiation. CONCLUSIONS: Downregulation of RAD9 when combined with ionizing radiation results in reduction of ITGB1 protein levels in prostate cancer cells, and increased lethality.

DNA damage response genes and the development of cancer metastasis.

DNA damage response genes play vital roles in the maintenance of a healthy genome. Defects in cell cycle checkpoint and DNA repair genes, especially mutation or aberrant downregulation, are associated with a wide spectrum of human disease, including a predisposition to the development of neurodegenerative conditions and cancer. On the other hand, upregulation of DNA damage response and repair genes can also cause cancer, as well as increase resistance of cancer cells to DNA damaging therapy. In recent years, it has become evident that many of the genes involved in DNA damage repair have additional roles in tumorigenesis, most prominently by acting as transcriptional (co-)factors. Although defects in these genes are causally connected to tumor initiation, their role in tumor progression is more controversial and it seems to depend on tumor type. In some tumors like melanoma, cell cycle checkpoint/DNA repair gene upregulation is associated with tumor metastasis, whereas in a number of other cancers the opposite has been observed. Several genes that participate in the DNA damage response, such as RAD9, PARP1, BRCA1, ATM and TP53 have been associated with metastasis by a number of in vitro biochemical and cellular assays, by examining human tumor specimens by immunohistochemistry or by DNA genome-wide gene expression profiling. Many of these genes act as transcriptional effectors to regulate other genes implicated in the pathogenesis of cancer. Furthermore, they are aberrantly expressed in numerous human tumors and are causally related to tumorigenesis. However, whether the DNA damage repair function of these genes is required to promote metastasis or another activity is responsible (e.g., transcription control) has not been determined. Importantly, despite some compelling in vitro evidence, investigations are still needed to demonstrate the role of cell cycle checkpoint and DNA repair genes in regulating metastatic phenotypes in vivo.