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.

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.

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.

Evaluation of a Device Combining an Inferior Vena Cava Filter and a Central Venous Catheter for Preventing Pulmonary Embolism Among Critically Ill Trauma Patients.

PURPOSE: To evaluate efficacy and safety of a novel device that combines an inferior vena cava (IVC) filter and central venous catheter (CVC) for prevention of pulmonary embolism (PE) in critically ill patients. MATERIALS AND METHODS: In a multicenter, prospective, single-arm clinical trial, the device was inserted at the bedside without fluoroscopy and subsequently retrieved before transfer from the intensive care unit (ICU). The primary efficacy endpoint was freedom from clinically significant PE or fatal PE 72 hours after device removal or discharge, whichever occurred first. Secondary endpoints were incidence of acute proximal deep venous thrombosis (DVT), catheter-related thrombosis, catheter-related bloodstream infections, major bleeding events, and clinically significant thrombus (occupying > 25% of volume of filter) detected by cavography before retrieval. RESULTS: The device was placed in 163 critically ill patients with contraindications to anticoagulation; 151 (93%) were critically ill trauma patients, 129 (85%) had head or spine trauma, and 102 (79%) had intracranial bleeding. The primary efficacy endpoint was achieved for all 163 (100%) patients (95% confidence interval [CI], 97.8%-100%, P < .01). Diagnosis of new or worsening acute proximal DVT was time dependent with 11 (7%) occurring during the first 7 days. There were no (0%) catheter-related bloodstream infections. There were 5 (3.1%) major bleeding events. Significant thrombus in the IVC filter occurred in 14 (8.6%) patients. Prophylactic anticoagulation was not initiated for a mean of 5.5 days ± 4.3 after ICU admission. CONCLUSIONS: This novel device prevented clinically significant and fatal PE among critically ill trauma patients with low risk of complications.

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.

The DNA damage checkpoint protein RAD9A is essential for male meiosis in the mouse.

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 IIß-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 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.

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.

PARP inhibition selectively increases sensitivity to cisplatin in ERCC1-low non-small cell lung cancer cells.

Platinum compounds are the foundation of chemotherapy regimens for non-small cell lung cancer (NSCLC) despite poor response rates and limited response duration. It has been reported that tumor expression of excision repair cross-complementation group 1 (ERCC1), a key component in nucleotide excision repair, may correlate with clinical response to platinum agents. We found that most primary lung tumor specimens demonstrated a stronger protein expression of poly (adenosine diphosphate ribose) polymerases 1 (PARP1) than their normal counterparts. Therefore, we hypothesized that combining PARP inhibition with platinum compounds may be an approach to improve platinum-based therapy for NSCLC. Drug combination experiments revealed that two distinct PARP inhibitors, olaparib and veliparib, not only potentiated the cell killing by cisplatin but also conferred cytotoxicity as a single agent specifically in ERCC1-low HCC827 and PC9 but not in ERCC1-high A549 and H157 lung cancer cells. Moreover, small interfering RNA knockdown of ERCC1 in A549 and H157 cells increased their sensitivities to both cisplatin and olaparib in a synergistic manner in our model. Furthermore, mechanistic studies indicated that combined PARP inhibitor and cisplatin could lead to sustained DNA double-strand breaks, prolonged G2/M cell cycle arrest with distinct activation of checkpoint kinase 1 signaling and more pronounced apoptosis preferentially in lung cancer cells with low ERCC1 expression. Collectively, these data suggest that there is a synergistic relationship between PARP inhibition and low ERCC1 expression in NSCLC that could be exploited for novel therapeutic approaches in lung cancer therapy based on tumor ERCC1 expression.

Rad9 protein contributes to prostate tumor progression by promoting cell migration and anoikis resistance.

Rad9 as part of the Rad9-Hus1-Rad1 complex is known to participate in cell cycle checkpoint activation and DNA repair. However, Rad9 can act as a sequence-specific transcription factor, modulating expression of a number of genes. Importantly, Rad9 is up-regulated in prostate cancer cell lines and clinical specimens. Its expression correlates positively with advanced stage tumors and its down-regulation reduces tumor burden in mice. We show here that transient down-regulation of Rad9 by RNA interference reduces DU145 and PC3 prostate cancer cell proliferation and survival in vitro. In addition, transient or stable down-regulation of Rad9 impairs migration and invasion of the cells. Moreover, stable reduction of Rad9 renders DU145 cell growth anchorage-dependent. It also decreases expression of integrin ß1 protein and sensitizes DU145 and LNCaP cells to anoikis and impairs Akt activation. On the other hand, stable expression of Mrad9, the mouse homolog, in DU145/shRNA Rad9 cells restores migration, invasion, anchorage-independent growth, integrin ß1 expression, and anoikis resistance with a concomitant elevation of Akt activation. We thus demonstrate for the first time that Rad9 contributes to prostate tumorigenesis by increasing not only tumor proliferation and survival but also tumor migration and invasion, anoikis resistance, and anchorage-independent growth.

Examining the Definition of Ventilator-Associated Pneumonia in the Trauma Setting: A Single-Center Analysis.

Background: Ventilator associated pneumonia (VAP) is defined by the American College of Surgeons Trauma Quality Improvement Program (ACS TQIP) using laboratory findings, pathophysiologic signs/symptoms, and imaging criteria. However, many critically ill trauma patients meet the non-specific laboratory and sign/symptom thresholds for VAP, so the TQIP designation of VAP depends heavily upon imaging evidence. We hypothesized that physician opinions widely vary regarding chest radiograph findings significant for VAP. Patients and Methods: The TQIP Spring 2021 Benchmark Report (BR) was used to identify 14 patients with VAP at an academic Level 1 Trauma Center. Critically ill trauma patients (n = 7) who spent at least four days intubated and met TQIP's laboratory and sign/symptom thresholds for VAP but did not appear as VAPs on the BR comprised the control group. For each deidentified patient, four successive chest radiographic images were compiled and arranged chronologically. Cases and controls were randomly arranged in digital format. Blinded physicians (n = 27) were asked to identify patients with VAP based solely on imaging evidence. Results: Radiographic evidence of VAP was highly subjective (Krippendorff α = 0.134). Among physicians of the same job description, inter-rater reliability remained low (α = 0.137 for trauma attending physicians; α = 0.141 for trauma fellows; α = 0.271 for radiologists). When majority judgment was compared to the TQIP BR, there was disagreement between the two tests (Cohen κ = -0.071; sensitivity, 64.3%; specificity, 28.6%). Conclusions: Current definitions of VAP rely on subjective imaging interpretation and ignore the reality that there are numerous explanations for opacities on CXR. The inconsistency of physicians' imaging interpretation and protean physiologic findings for VAP in trauma patients should preclude the current definition of VAP from being used as a quality improvement metric in TQIP.

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.

Contributions of Rad9 to tumorigenesis.

Rad9 plays a crucial role in maintaining genomic stability by regulating cell cycle checkpoints, DNA repair, telomere stability, and apoptosis. Rad9 controls these processes mainly as part of the heterotrimeric 9-1-1 (Rad9-Hus1-Rad1) complex. However, in recent years it has been demonstrated that Rad9 can also act independently of the 9-1-1 complex as a transcriptional factor, participate in immunoglobulin class switch recombination, and show 3'-5' exonuclease activity. Aberrant Rad9 expression has been associated with prostate, breast, lung, skin, thyroid, and gastric cancers. High expression of Rad9 is causally related to, at least, human prostate cancer growth. On the other hand, deletion of Mrad9, the mouse homolog, is responsible for increased skin cancer incidence. These results reveal that Rad9 can act as an oncogene or tumor suppressor. Which of the many functions of Rad9 are causally related to initiation and progression of tumorigenesis and the mechanistic details by which Rad9 induces or suppresses tumorigenesis are presently not known, but are crucial for the development of targeted therapeutic interventions.

Mouse Rad9b is essential for embryonic development and promotes resistance to DNA damage.

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.

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.

Acute Kidney Injury Risk in Patients Treated with Vancomycin Combined with Meropenem or Cefepime.

Background: No previous studies have determined the incidence of acute kidney injury (AKI) in trauma patients treated with vancomycin + meropenem (VM) versus vancomycin + cefepime (VC). The purpose of this study was to fill this gap. Methods: A series of 99 patients admitted to an American College of Surgeons-verified level 1 trauma center over a two-year period who received VC or VM for >48 hours were reviewed retrospectively. Exclusion criteria were existing renal dysfunction or on renal replacement therapy. The primary outcome was AKI as defined by a rise in serum creatinine (SCr) to 1.5 times baseline. Multi-variable analysis was performed to control for factors associated with AKI (age, obesity, gender, length of stay [LOS], nephrotoxic agent(s), and baseline SCr), with significance defined as p < 0.05. Results: The study population was 50 ± 19 years old, 76% male, with a median LOS of 21 [range 15-39] days, and baseline SCr of 0.9 ± 0.2 mg/dL. Antibiotics, diabetes mellitus, and Injury Severity Score were independent predictors of AKI (odds ratio [OR] 4.4; 95% confidence interval [CI] 1.4-12; OR 9.3; 95% CI 1-27; OR 1.2; 95% CI 1.023-1.985, respectively). The incidence of AKI was higher with VM than VC (10/26 [38%] versus 14/73 [19.1%]; p = 0.049). Conclusions: The renal toxicity of vancomycin is potentiated by meropenem relative to cefepime in trauma patients. We recommend caution when initiating vancomycin combination therapy, particularly with meropenem.

Mouse Rad1 deletion enhances susceptibility for skin tumor development.

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.

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.

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.

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.