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Decreased E-cadherin immunostaining is frequently observed in benign prostatic hyperplasia (BPH) and was recently correlated with increased inflammation in aging prostate. Homozygous E-cadherin deletion in the murine prostate results in prostate inflammation and bladder overactivity at 6 months of age. However, this model is limited in that while E-cadherin is significantly reduced in BPH, it is not completely lost; BPH is also strongly associated with advanced age and is infrequent in young men. Here, we examined the functional consequences of aging in male mice with prostate luminal epithelial cell-specific E-cadherin heterozygosity. In control mice, aging alone resulted in an increase in prostate inflammation and changes in bladder voiding function indicative of bladder underactivity. At 24 months of age, mice with prostate-specific Cre-mediated heterozygous deletion of E-cadherin induced at 7 weeks of age developed additional prostatic defects, particularly increased macrophage inflammation and stromal proliferation, and bladder overactivity compared to age-matched control mice, which are similar to BPH/LUTS in that the phenotype is slow-progressing and age-dependent. These findings suggest that decreased E-cadherin may promote macrophage inflammation and fibrosis in the prostate and subsequent bladder overactivity in aging men, promoting the development and progression of BPH/LUTS.

4E-BP1 is a tumor suppressor regulating cap-dependent translation that is in turn controlled by mechanistic target of rapamycin (mTOR) or cyclin-dependent kinase 1 (CDK1) phosphorylation. 4E-BP1 serine 82 (S82) is phosphorylated by CDK1, but not mTOR, and the consequences of this mitosis-specific phosphorylation are unknown. Knock-in mice were generated with a single 4E-BP1 S82 alanine (S82A) substitution leaving other phosphorylation sites intact. S82A mice were fertile and exhibited no gross developmental or behavioral abnormalities, but the homozygotes developed diffuse and severe polycystic liver and kidney disease with aging, and lymphoid malignancies after irradiation. Sublethal irradiation caused immature T-cell lymphoma only in S82A mice while S82A homozygous mice have normal T-cell hematopoiesis before irradiation. Whole genome sequencing identified PTEN mutations in S82A lymphoma and impaired PTEN expression was verified in S82A lymphomas derived cell lines. Our study suggests that the absence of 4E-BP1S82 phosphorylation, a subtle change in 4E-BP1 phosphorylation, might predispose to polycystic proliferative disease and lymphoma under certain stressful circumstances, such as aging and irradiation.

Early detection of lung cancer is essential to reduce lung cancer-related morbidity and mortality. Despite significant efforts, current approaches suffer from high false-positive rates or limited sensitivity. There is a critical need for non-invasive, cost-effective technologies that detect the early changes in lung carcinogenesis with high sensitivity and specificity. Abnormal chromatin structure is one of the hallmarks of cancer cells and is independent of molecular pathways making it an attractive target for early cancer detection. In this study, we assessed the use of super-resolution stochastic optical reconstruction microscopy (STORM) to detect abnormal heterochromatin disruption in lung carcinogenesis. First, utilizing a mouse model of tobacco carcinogen (NNK)-induced lung adenocarcinoma, we analyzed heterochromatin structure in the lungs of NNK-treated mice at 8, 15 and 20 weeks post-NNK exposure together with non-treated control mice at the same time points. Super-resolution images of NNK-induced lung lesions at all time points showed significant disruption of condensed heterochromatin nanodomains compared to normal alveolar epithelial cells from the control mice, consistent with our data from other tumor types. Importantly, the normal-appearing bronchial epithelial cell nuclei from NNK-treated mice also showed significant heterochromatin disruption in NNK-treated mice at 15 and 20 weeks (P< 0.0001), demonstrating that this method can detect early changes. Next, we sought to confirm this in humans. Super-resolution imaging showed that, compared to normal alveolar and bronchial epithelial cells from control subjects without cancer, the heterochromatin structure was more disrupted in tumor cells from patients with lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). We also observed significant disrupted heterochromatin structure in tumor-adjacent normal tissue at the tumor margin. Lastly, we performed a pilot study on bronchial epithelial cells isolated from sputum samples from 11 cigarette smokers with or without lung cancer. The heterochromatin structure in the bronchial epithelial cells collected from lung cancer patients at the time of diagnosis was significantly more disrupted than those from smokers without lung cancer (P= 0.004). Our results support disrupted heterochromatin structure as an early event in lung carcinogenesis and demonstrate the feasibility of utilizing super-resolution imaging of heterochromatin structure for early detection of lung cancer using sputum samples. Citation Format: Laura P. Stabile, Xuejiao Sun, Jianquan Xu, Beatriz Kanterewicz, David O. Wilson, Sanja Dacic, Lora H. Rigatti, Brenda Diergaarde, Yang Liu. Super-resolution imaging of heterochromatin structure detects early events in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 777.

Cancer is often treated with DNA-damaging cytotoxic and ionizing radiation (IR) therapies. However, anticancer efficacy is limited by activation of DNA damage response (DDR) pathways that support DNA repair resulting in cancer cell survival. Disruption of DDR pathways may enhance cancer cell sensitivity to DNA-damaging therapies. Ataxia telangiectasia and Rad3 related (ATR) protein kinase, an apical initiator of DDR, is highly relied on by tumor cells and is critical for survival. Pharmacological ATR inhibitors (ATRi) can sensitize cancer cells to DNA damaging therapies. Although combining radiation therapies with ATRi may enhance anticancer efficacy, the resultant toxicity to normal organs is largely unknown. The ATRi furthest in development are the orally administered AZD6738 (ceralasertib), BAY-1895344 (elimusertib) and the intravenously administered M6620 (berzosertib, VX-970). We aimed to characterize and compare the preclinical toxicity of ATRi with and without total body irradiation (TBI) by pathology and complete blood counts (CBC). To determine the appropriate TBI dose for combination with ATRi, female balb/c mice (n=6) were grouped to receive 2, 4, 6 or 8 Gy. Within these cohorts, mice (n=2) were subdivided for euthanization times of 48, 72 and 96 h post TBI. Next, mice (N=5) were treated with a single dose of ATRi, 6 Gy TBI, the combination (TBI within 30 min of ATRi), or control. Mice were euthanized 48 h post treatment and blood was collected for analysis of CBC. Changes to body weight were documented and tissues with known sensitivities to radiation-related toxicity were collected for histopathology. Statistical analyses were performed using ANOVA with Tukey’s multiple comparisons test. A 6 Gy TBI dose and 48 h euthanasia time point were selected for investigation in combination studies with ATRi. TBI alone resulted in ~5% decreased body weight compared to vehicle controls. The addition of any ATRi appeared to ameliorate this loss, but not to a significant extent. TBI caused leukocytopenia and lymphocytopenia, which was not exacerbated by the addition of any ATRi. Neutrophilia was observed after ATRi alone and abolished by addition of TBI. TBI caused moderate toxicity in spleen, small intestine and bone marrow, and the addition of ATRi did not exacerbate these findings. AZD6738 caused moderate pericarditis and myocarditis, which was abrogated by the addition of IR. Collectively, ATRi administered alone are associated with neutrophilia at 48 h. AZD6738 appeared to be associated with cardiac toxicity, consistent with clinical observations of related CHK1 inhibitors. M6620 alone is associated with a significant decrease in lymphocytes compared to vehicle control, however in the setting of TBI, M6620 appears to ameliorate TBI-induced lymphocytopenia. This study demonstrated that the addition of ATRi did not increase toxicity associated with TBI in mice. Citation Format: Joshua J. Deppas, Brian F. Kiesel, Jianxia Guo, Lora H. Rigatti, Joseph Latoche, Anthony Green, Paul Knizner, Christopher J. Bakkenist, Jan H. Beumer. Toxicological characterization and comparison of ATR inhibitors in mice. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5061.

Clostridium difficile is a well-documented cause of enterocolitis in several species, including humans, with limited documentation in New World nonhuman primates. We report several cases of C. difficile–associated pseudomembranous enterocolitis, including a case in a Geoffroy’s spider monkey ( Ateles geoffroyi) and several cases in common marmosets ( Callithrix jacchus). The histologic lesions included a spectrum of severity, with most cases characterized by the classic “volcano” lesions described in humans and several other animal species. C. difficile was isolated from the colon of the spider monkey, while the presence of toxin A or toxin B or of the genes of toxin A or B by polymerase chain reaction served as corroborative evidence in several affected marmosets. C. difficile should be considered a cause of enterocolitis in these species.

PURPOSE: Prostate-specific membrane antigen (PSMA) continues to be the hallmark biomarker for prostate cancer as it is expressed on nearly all prostatic tumors. In addition, increased PSMA expression correlates with castration resistance and progression to the metastatic stage of the disease. Recently, we combined both an albumin-binding motif and an irreversible PSMA inhibitor to develop the novel PSMA-targeted radiotherapeutic agent, CTT1403. This molecule was novel in the field of PSMA-targeted agents as its key motifs resulted in extended blood circulation time and tumor uptake, rapid and extensive internalization into PSMA+ cells, and promising therapeutic efficacy. The objective of this study was to perform IND-enabling translational studies on CTT1403 in rodent models. PROCEDURES: A dose optimization study was performed in PSMA+ tumor-bearing mice. Treatment groups were randomly selected to receive one to three 14 MBq injections of CTT1403. Control groups included: 1) saline; 2) non-radioactive [(175)Lu]CTT1403; or 3) two injections of 14 MBq CTT1751, a Lu-177 labeled non-targeted radiolabeled albumin-binding moiety. Tumor growth was monitored up to 120 days. Small animal single photon emission tomography/x-ray computed tomography imaging was performed with CTT1403 and CTT1751 in PC3-PIP tumor-bearing mice to visualize infiltration of the Lu-177 labeled agent into the tumor. In preparation for a first-in-human study, human absorbed doses were estimated based on rat biodistribution out to 5 weeks to determine a safe CTT1403 therapy dose in humans. RESULTS: Two to 3 injections of 14 MBq CTT1403 yielded significant tumor growth inhibition and increased survival compared to all control groups and mice receiving 1 injection of 14 MBq CTT1403. Five of 12 mice receiving 2 or 3 injections of CTT1403 survived to the 120 days post-treatment study endpoint. Dosimetry identified the kidneys as the dose limiting organ, with an equivalent dose of 5.18 mSv/MBq, resulting in a planned maximum dose of 4.4 GBq for Phase 1 clinical trials. CONCLUSIONS: The preclinical efficacy and dosimetry of CTT1403 suggest that this agent has significant potential to be safe and effective in humans.

Aim To demonstrate that Fanconi anemia complementation group D2-deficient (Fancd2-/-) hematopoietic progenitor cell lines can be transformed by transfection with a plasmid containing either the E6 or E7 oncogene of human papillomavirus (HPV) to generate malignant plasmacytoma-inducing cell lines. Materials and methods In order to determine whether a single HPV type 16 (HPV16) oncogene induced malignant transformation, Fancd2-/- and Fancd2+/+ interleukin 3 (IL3)-dependent hematopoietic progenitor cell lines were transfected with plasmids containing E6 or E7 oncogene, or control empty plasmid. Results Fancd2-/- but not Fancd2+/+ cells were transformed into malignant IL3-independent cells by both E6, and E7 oncogenes, but not by empty plasmid. Hematopoietic cell lines and tumors induced by Fancd2-/- E6 and Fancd2-/- E7 cell lines were positive for each respective HPV RNA and protein. Conclusion A single HPV16 oncogene is adequate to produce malignant transformation of Fancd2-/- hematopoietic cells.

Benign prostatic hyperplasia (BPH) is an age-related debilitating prostatic disease that is frequently associated with prostatic inflammation and bothersome lower urinary tract symptoms (LUTS). Animal models have shown that formalin- and bacterial-induced prostatic inflammation can induce bladder dysfunction; however, the underlying mechanisms contributing to prostatic inflammation in BPH and bladder dysfunction are not clear. We previously reported that E-cadherin expression in BPH is downregulated in hyperplastic nodules compared with expression in adjacent normal tissues. Here, we explored the potential consequences of prostatic E-cadherin downregulation on the prostate and bladder in vivo using an inducible murine model of prostate luminal epithelial-specific deletion of Cdh1. The prostate-specific antigen (PSA)-CreERT2 transgenic mouse strain expressing tamoxifen-inducible CreERT2 recombinase driven by a 6-kb human PSA promoter/enhancer was crossed with the B6.129-Cdh1tm2Kem/J mouse to generate bigenic PSA-CreERT2/Cdh1-/- mice. Deletion of E-cadherin was induced by transient administration of tamoxifen when mice reached sexual maturity (7 weeks of age). At 21 to 23 weeks of age, the prostate, bladder, and prostatic urethra were examined histologically, and bladder function was assessed using void spot assays and cystometry. Mice with Cdh1 deletion had increased prostatic inflammation, prostatic epithelial hyperplasia, and stromal changes at 21 to 23 weeks of age, as well as changes in bladder voiding function compared with age-matched controls. Thus, loss of E-cadherin in the murine prostate could result in prostatic defects that are characteristic of BPH and LUTS, suggesting that E-cadherin downregulation could be a driving force in human BPH development and progression.

Develop a clinically relevant and reproducible endoscopic animal model for subglottic stenosis amenable to testing of minimally invasive therapeutic modalities.Cohort study.Division of Laboratory Animals Research, University of Pittsburgh.Subglottic stenosis was induced endoscopically via microsuspension laryngoscopy in 26 New Zealand white rabbits. A trimmed polypropylene brush connected to a novel electronic stenosis induction apparatus was used to create circumferential trauma to the subglottis. By using open source image analysis software, the cross-sectional areas of the stenotic and native airways were compared to calculate the percentage of stenosis and the Myer-Cotton classification grade.Of the 26 rabbits, 24 (92%) exhibited stenosis after the first attempt. The mean percentage of airway stenosis was 57% (range, 34%-85%; SD, 15%). Five rabbits (19.2%) died on the day of stenosis induction from procedural complications. Of the 21 rabbits, 2 demonstrated no stenosis 7 days after initial injury and so underwent reinduction of airway injury, upon which they developed stenosis. Overall, 14 of the 21 rabbits (67%) exhibited moderate to severe stenosis (grade 2 or 3).The stenosis induction apparatus reliably induced stenosis with a low mortality rate as compared with that of other methods in the literature. The device could be improved to generate a predetermined potentially reproducible grade of stenosis as desired by the operator. This method sets the stage for a clinically relevant and reproducible subglottic stenosis disease model that is amenable to testing of minimally invasive treatment modalities.

Abundant intraperitoneal (IP) accumulation of extracellular mucus in patients with appendiceal mucinous carcinoma peritonei (MCP) causes compressive organ dysfunction and prevents delivery of chemotherapeutic drugs to cancer cells. We hypothesized that reducing extracellular mucus would decrease tumor-related symptoms and improve chemotherapeutic effect in patient-derived models of MCP. Mucolysis was achieved using a combination of bromelain (BRO) and N-acetylcysteine (NAC). Ex vivo experiments of mucolysis and chemotherapeutic drug delivery/effect were conducted with MCP and non-MCP tissue explants. In vivo experiments were performed in mouse and rat patient-derived xenograft (PDX) models of early and late (advanced) MCP. MCP tumor explants were less chemosensitive than non-MCP explants. Chronic IP administration of BRO + NAC in a mouse PDX model of early MCP and a rat PDX model of late (advanced) MCP converted solid mucinous tumors into mucinous ascites (mucolysis) that could be drained via a percutaneous catheter (rat model only), significantly reduced solid mucinous tumor growth and improved the efficacy of chemotherapeutic drugs. Combination of BRO + NAC efficiently lyses extracellular mucus in clinically relevant models of MCP. Conversion of solid mucinous tumors into mucinous ascites decreases tumor bulk and allows for minimally invasive drainage of liquified tumors. Lysis of extracellular mucus removes the protective mucinous coating surrounding cancer cells and improves chemotherapeutic drug delivery/efficacacy in cancer cells. Our data provide a preclinical rationale for the clinical evaluation of BRO + NAC as a therapeutic strategy for MCP.

Lipid peroxidation (LPO) products are relatively stable and abundant metabolites, which accumulate in tissues of mammals with aging, being able to modify all cellular nucleophiles, creating protein and DNA adducts including crosslinks. Here, we used cells and mice deficient in the ERCC1-XPF endonuclease required for nucleotide excision repair and the repair of DNA interstrand crosslinks to ask if specifically LPO-induced DNA damage contributes to loss of cell and tissue homeostasis. Ercc1(−/−) mouse embryonic fibroblasts were more sensitive than wild-type (WT) cells to the LPO products: 4-hydroxy-2-nonenal (HNE), crotonaldehyde and malondialdehyde. ERCC1-XPF hypomorphic mice were hypersensitive to CCl(4) and a diet rich in polyunsaturated fatty acids, two potent inducers of endogenous LPO. To gain insight into the mechanism of how LPO influences DNA repair-deficient cells, we measured the impact of the major endogenous LPO product, HNE, on WT and Ercc1(−/−) cells. HNE inhibited proliferation, stimulated ROS and LPO formation, induced DNA base damage, strand breaks, error-prone translesion DNA synthesis and cellular senescence much more potently in Ercc1(−/−) cells than in DNA repair-competent control cells. HNE also deregulated base excision repair and energy production pathways. Our observations that ERCC1-deficient cells and mice are hypersensitive to LPO implicates LPO-induced DNA damage in contributing to cellular demise and tissue degeneration, notably even when the source of LPO is dietary polyunsaturated fats.

We have shown previously that dietary administration of phenethyl isothiocyanate (PEITC), a small molecule from edible cruciferous vegetables, significantly decreases the incidence of poorly differentiated prostate cancer in Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice without any side effects. In this study, we investigated the role of c-Myc–regulated glycolysis in prostate cancer chemoprevention by PEITC. Exposure of LNCaP (androgen-responsive) and 22Rv1 (castration-resistant) human prostate cancer cells to PEITC resulted in suppression of expression as well as transcriptional activity of c-Myc. Prostate cancer cell growth inhibition by PEITC was significantly attenuated by stable overexpression of c-Myc. Analysis of the RNA-Seq data from The Cancer Genome Atlas indicated a significant positive association between Myc expression and gene expression of many glycolysis-related genes, including hexokinase II and lactate dehydrogenase A. Expression of these enzyme proteins and lactate levels were decreased upon PEITC treatment in prostate cancer cells, and these effects were significantly attenuated by ectopic expression of c-Myc. A normal prostate stromal cell line (PrSC) was resistant to lactic acid suppression by PEITC treatment. Prostate cancer chemoprevention by PEITC in TRAMP mice was associated with a significant decrease in plasma lactate and pyruvate levels. However, a 1-week intervention with 10 mg PEITC (orally, 4 times/day) was not sufficient to decrease lactate levels in the serum of human subjects. These results indicated that although prostate cancer prevention by PEITC in TRAMP mice was associated with suppression of glycolysis, longer than 1-week intervention might be necessary to observe such an effect in human subjects. Cancer Prev Res; 11(6); 337–46. ©2018 AACR.

The tumor suppressor genes EAF2 and p53 are frequently dysregulated in prostate cancers. Recently, we reported that concurrent p53 nuclear staining and EAF2 downregulation were associated with high Gleason score. Combined loss of EAF2 and p53 in a murine model induced prostate tumors, and concurrent knockdown of EAF2 and p53 in prostate cancer cells enhanced proliferation and migration, further suggesting that EAF2 and p53 could functionally interact in the suppression of prostate tumorigenesis. Here, RNA-seq analyses identified differentially regulated genes in response to concurrent knockdown of p53 and EAF2. Several of these genes were associated with the STAT3 signaling pathway, and this was verified by significantly increased p-STAT3 immunostaining in the Eaf2−/−p53−/− mouse prostate. STAT3 knockdown abrogated the stimulation of C4-2 cell proliferation by concurrent knockdown of EAF2 and p53. Furthermore, immunostaining of p-STAT3 was increased in human prostate cancer specimens with EAF2 downregulation and/or p53 nuclear staining. Our findings suggest that simultaneous inactivation of EAF2 and p53 can act to activate STAT3 and drive prostate tumorigenesis.

Aims Respiratory disorders are a prominent component of Gulf War Illness. Although much of the underlying mechanisms of Gulf War Illness remain undefined, chronic immune dysfunction is a consistent feature of this multi-symptomatic, multi-organ disorder. Alveolar macrophages represent the predominant mononuclear phagocytes of the pulmonary mucosa, orchestrating the host response to pathogens and environmental stimuli. Herein, we sought to characterize the innate immune response of the pulmonary mucosa, with a focus on macrophages, to experimental respiratory exposure to two putative Gulf War Toxins (GWTs). Materials and methods Utilizing commercially available instrumentation, we evaluated the effect of aerosolized exposure to the pesticide malathion and diesel exhaust particulate (DEP) on the immune composition and inflammatory response of the lung in FVB/N mice using multiparametric spectral cytometry, cytokine analysis, and histology. Key findings Aerosolized GWTs induced gross pulmonary pathology with transient recruitment of neutrophils and sustained accumulation of alveolar macrophages to the lung for up to two weeks after exposure cessation. High-dimensional cytometry and unbiased computational analysis identified novel myeloid subsets recruited to the lung post-exposure driven by an influx of peripheral monocyte-derived progenitors. DEP and malathion, either alone or in combination, induced soluble mediators in bronchoalveolar lavage indicative of oxidative stress (PGF2α), inflammation (LTB4, TNFα, IL-12), and immunosuppression (IL-10), that were sustained or increased two weeks after exposures concluded. Significance These findings indicate that macrophage accumulation and pulmonary inflammation induced by GWTs continue in the absence of toxin exposure and may contribute to the immunopathology of respiratory Gulf War Illness.

Respiratory disorders including chronic obstructive pulmonary disease, asthma, and increased susceptibility to lung infections are a prominent component of Gulf War Illness (GWI). Although the mechanisms of GWI are unknown, chronic immune dysfunction is a consistent factor of this multi-symptomatic disorder. Alveolar macrophages (AMs) represent the predominant mononuclear phagocytes of the pulmonary mucosa, orchestrating the host response to pathogens and environmental stimuli via balanced induction of inflammatory, anti-inflammatory, and pro-resolution mediators. Utilizing commercially available instrumentation, we evaluated the effect of aerosolized exposure to two putative GW toxins, the pesticide malathion and diesel exhaust particulate (DEP), on the immune composition and inflammatory response of the lung in FVB/N mice. Toxin exposure resulted in gross lung pathology with variable increases in protein, lactose dehydrogenase, and cytokines in bronchoalveolar lavage (BAL). Aerosolized toxin exposure reduced AMs in the lung and BAL, associated with a shift to wound-healing phenotypes and concomitant influx of monocyte-derived progenitors from the circulation. Exposure to DEP and malathion, either alone or in combination, induced varying levels of dysfunction in flow-sorted AMs ex vivo as determined by inhibited migration, cytokine production, and phagocytosis. Toxin-induced alterations were associated with unique transcriptional profiles following RNA sequencing analysis. Collectively, these findings suggest that respiratory toxin-induced macrophage dysfunction may establish a state of frustrated immune resolution underlying the chronic immunopathology of respiratory GWI.

Mutations in the p53 tumor suppressor are frequent in patients with castration-resistant prostate cancer but less so in patients with localized disease, and patients who have Li-Fraumeni with germline p53 mutations do not have an increased incidence of prostate cancer, suggesting that additional molecular and/or genetic changes are required for p53 to promote prostate carcinogenesis. ELL-associated factor 2 (EAF2) is a tumor suppressor that is frequently downregulated in advanced prostate cancer. Previous studies have suggested that p53 binds to EAF2, providing a potential mechanism for their functional interactions. In this study, we tested whether p53 and EAF2 could functionally interact in prostate cancer cells and whether concurrent inactivation of p53 and EAF2 could promote prostate carcinogenesis in a murine knockout model. Endogenous p53 coprecipitated with EAF2 in prostate cancer cells, and deletion mutagenesis indicated that this interaction was mediated through the C terminus of EAF2 and the DNA binding domain of p53. Concurrent knockdown of p53 and EAF2 induced an increase in proliferation and migration in cultured prostate cancer cells, and conventional p53 and EAF2 knockout mice developed prostate cancer. In human prostate cancer specimens, concurrent p53 nuclear staining and EAF2 downregulation was associated with high Gleason score. These findings suggest that EAF2 and p53 functionally interact in prostate tumor suppression and that simultaneous inactivation of EAF2 and p53 can drive prostate carcinogenesis.

Background/Aim: The cause of fatal neuromuscular amyotrophic lateral sclerosis (ALS) is not known. Materials and Methods: Ninety-day-old superoxide-dismutase-1(G93A) (SOD1(G93A)) mice demonstrating level 1 paralysis, received 9.0 Gy total body irradiation (TBI) from a cesium source at 340 cGy per minute, and intravenous transplantation with 1×10(6) C57BL/6 green fluorescent protein (GFP)+ donor bone marrow cells. Results: Paralysis-free survival was prolonged in TBI and bone marrow-transplanted SOD1(G93A) mice from 100 to over 250 days (p=0.0018). Other mice transplanted with SOD1(G93A) marrow or marrow treated with the free-radical scavenger MMS350 showed no therapeutic effect. GFP+ macrophage-2 (M(2)) microglial cells of bone marrow origin, were seen at sites of degenerating anterior horn motor neurons. SOD1(G93A) mice had a disruption in the blood–brain barrier permeability which was reversed by marrow transplant from C57BL/6 mice. SOD1(G93A) marrow showed unexpected robust hematopoiesis in vitro, and radioresistance. Conclusion: After TBI, M(2) microglial cells from transplanted donor marrow extended the paralysis-free interval in SOD1(G93A) mice.

ELL-associated factor 1 is a transcription elongation factor that shares significant homology and functional similarity to the androgen-responsive prostate tumor suppressor ELL-associated factor 2. EAF2 is frequently down-regulated in advanced prostate cancer and Eaf2 deletion in the mouse induced the development of murine prostatic intraepithelial neoplasia. Here we show that similar to EAF2, EAF1 is frequently down-regulated in advanced prostate cancer. Co-downregulation of EAF1 and EAF2 occurred in 40% of clinical specimens with Gleason score >7. We developed and characterized a murine model of prostate-epithelial specific deletion of Eaf1 in the prostate and crossed it with our previously generated mouse with conventional deletion of Eaf2. The prostates of Eaf1 deletion mice displayed murine prostatic intraepithelial neoplasia lesions with increased proliferation and inflammation. Combined deletion of Eaf1 and Eaf2 in the murine model induced an increased incidence in mPIN lesions characterized by increased proliferation and CD3+ T cells and CD19+ B cells infiltration compared to individual deletion of either Eaf1 or Eaf2 in the murine prostate. These results suggest that EAF1 may play a tumor suppressive role in the prostate. Cooperation between EAF1 and EAF2 may be important for prostate maintaining prostate epithelial homeostasis, and concurrent loss of these two tumor suppressors may promote prostate tumorigenesis and progression.

// Xichen Zhang 1 , Wen Hou 1 , Michael W. Epperly 1 , Lora Rigatti 2 , Hong Wang 1 , Darcy Franicola 1 , Aranee Sivanathan 1 , Joel S. Greenberger 1 1 Department of Radiation Oncology, University of Pittsburgh Cancer Institute, Pittsburgh, 15232 PA, USA 2 Division of Laboratory Animal Resources, University of Pittsburgh, Pittsburgh, 15260 PA, USA Correspondence to: Joel S. Greenberger, email: greenbergerjs@upmc.edu Keywords: E7, HPV oncogene, human papillomavirus, Fanconi anemia, Fancd2 −/− mice Received: April 29, 2016 Accepted: September 02, 2016 Published: September 15, 2016 ABSTRACT We tested the effect of expression of the Human Papilloma Virus (HPV E7) oncogene on hematopoiesis in long-term bone marrow cultures (LTBMCs) derived from K14E7 (FVB) Fancd2 −/− (129/Sv), K14E7 Fancd2 +/+ , Fancd2 −/− , and control (FVB X 129/Sv) Fl mice. K14E7 Fancd2 −/− and Fancd2 −/− LTBMCs showed decreased duration of production of total nonadherent hematopoietic cells and progenitors forming day 7 and day 14 multilineage CFU-GEMM colonies in secondary cultures (7 wks and 8 wks respectively) compared to cultures from K14E7 Fancd2 +/+ (17 wks) or control mice (18 wks) p < 0.0001. Marrow stromal cell lines derived from both K14E7 Fancd2 −/− and Fancd2 −/− cultures were radiosensitive, as were IL-3 dependent hematopoietic progenitor cell lines derived from K14E7 Fancd2 −/− cultures. In contrast, Fancd2 −/− mouse hematopoietic progenitor cell lines and fresh marrow were radioresistant. K14E7 Fancd2 −/− mouse freshly explanted bone marrow expressed no detectable K14 or E7; however, LTBMCs produced K14 positive factor-independent (FI) clonal malignant plasmacytoma forming cell lines in which E7 was detected in the nucleus with p53 and Rb. Transfection of an E6/E7 plasmid into Fancd2 −/− , but not control Fancd2 +/+ IL-3 dependent hematopoietic progenitor cell lines, increased cloning efficiency, cell growth, and induced malignant cell lines. Therefore, the altered radiobiology of hematopoietic progenitor cells and malignant transformation in vitro by K14E7 expression in cells of the Fancd2 −/− genotype suggests a potential role of HPV in hematopoietic malignancies in FA patients.

The present study is the first to report inhibition of breast cancer cell growth in vitro and in vivo and suppression of self-renewal of breast cancer stem cells (bCSC) by a pine bark component (leelamine). Except for a few recent publications in melanoma, anticancer pharmacology of this interesting phytochemical is largely elusive. Leelamine (LLM) dose-dependently inhibited viability of MDA-MB-231 (triple-negative), MCF-7 (estrogen receptor-positive), and SUM159 (triple-negative) human breast cancer cells in association with apoptotic cell death induction. To the contrary, a normal mammary epithelial cell line derived from fibrocystic breast disease and spontaneously immortalized (MCF-10A) was fully resistant to LLM-mediated cell growth inhibition and apoptosis induction. LLM also inhibited self-renewal of breast cancer stem cells. Apoptosis induction by LLM in breast cancer cells was accompanied by a modest increase in reactive oxygen species production, which was not due to inhibition of mitochondrial electron transport chain complexes. Nevertheless, ectopic expression of manganese superoxide dismutase conferred partial protection against LLM-induced cell death but only at a lower yet pharmacologically relevant concentration. Exposure of breast cancer cells to LLM resulted in (a) induction and/or activation of multidomain proapoptotic proteins Bax and Bak, (b) caspase-9 activation, and (c) cytosolic release of cytochrome c. Bax and Bak deficiency in immortalized fibroblasts conferred significant protection against cell death by LLM. Intraperitoneal administration of LLM (7.5 mg/kg; 5 times/wk) suppressed the growth of orthotopic SUM159 xenografts in mice without any toxicity. In conclusion, the present study provides critical preclinical data to warrant further investigation of LLM. © 2016 Wiley Periodicals, Inc.

Elongation factor for RNA polymerase II 2 (ELL2) and ELL-associated factor 2 (EAF2) are two functionally related androgen responsive gene-encoded proteins with prostate tumor suppressor characteristics. EAF2 and ELL2 have both been shown to be down-regulated in advanced prostate cancer, and mice with either Eaf2 or Ell2 deficiency developed murine prostatic intraepithelial neoplasia (mPIN), increased cellular proliferation and increased vascularity. Functional studies have revealed that EAF2 and ELL2 can bind to each other and have similar roles in regulating cell proliferation, angiogenesis and prostate homeostasis. Here, cell line experiments showed that knockdown of EAF2 or ELL2 induced an increase in proliferation and migration in C4-2 and 22Rv1 prostate cancer cells. Concurrent knockdown of EAF2 and ELL2 increased proliferation and migration similarly to the loss of EAF2 or ELL2 alone. Mice with homozygous deletion of Ell2 or heterozygous deletion of Eaf2 developed mPIN lesions characterized by increased epithelial proliferation, intraductal microvessel density, and infiltrating intraductal CD3-positive T-cells compared to wild-type controls. Mice with combined heterozygous deletion of Eaf2 and Ell2 developed mPIN lesions that were similar to those observed in mice with deficiency in Eaf2 or Ell2 alone. These results suggest that EAF2 and ELL2 have similar functions and are likely to require each other in their regulation of prostate epithelial cell proliferation and migration in prostate cancer cells as well as their tumor suppressive properties in the murine prostate.

Accumulation of senescent cells over time contributes to aging and age-related diseases. However, what drives senescence in vivo is not clear. Here we used a genetic approach to determine if spontaneous nuclear DNA damage is sufficient to initiate senescence in mammals. Ercc1-/∆ mice with reduced expression of ERCC1-XPF endonuclease have impaired capacity to repair the nuclear genome. Ercc1-/∆ mice accumulated spontaneous, oxidative DNA damage more rapidly than wild-type (WT) mice. As a consequence, senescent cells accumulated more rapidly in Ercc1-/∆ mice compared to repair-competent animals. However, the levels of DNA damage and senescent cells in Ercc1-/∆ mice never exceeded that observed in old WT mice. Surprisingly, levels of reactive oxygen species (ROS) were increased in tissues of Ercc1-/∆ mice to an extent identical to naturally-aged WT mice. Increased enzymatic production of ROS and decreased antioxidants contributed to the elevation in oxidative stress in both Ercc1-/∆ and aged WT mice. Chronic treatment of Ercc1-/∆ mice with the mitochondrial-targeted radical scavenger XJB-5–131 attenuated oxidative DNA damage, senescence and age-related pathology. Our findings indicate that nuclear genotoxic stress arises, at least in part, due to mitochondrial-derived ROS, and this spontaneous DNA damage is sufficient to drive increased levels of ROS, cellular senescence, and the consequent age-related physiological decline. Keywords: Reactive oxygen species, Free radicals, Genotoxic stress, Oxidative lesions, Endogenous DNA damage, Cellular senescence, Aging

Background Pericytes have been shown to have mesenchymal stromal cell–like properties and play a role in tissue regeneration. The goal of this study was to determine whether the addition of a pericyte sheet to a full-thickness dermal wound would enhance the healing of an acute wound. Methods Human muscle-derived pericytes and human dermal fibroblasts were formed into cell sheets, then applied to full-thickness excisional wounds on the dorsum of nu/nu mice. Histology was performed to evaluate epidermal and dermal reformation, inflammation and fibrosis. In addition, real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was used to determine cytokine response. Results Pericytes were detected in the wounds until day 16 but not fibroblasts. Decrease in wound size was noted in pericyte sheet-treated wounds. Enhanced neo-vascularization and healthy granulation tissue formation were noted in the pericyte-treated wounds. Expression of type I collagen messenger RNA (mRNA) was significantly higher in the fibroblast-treated group, whereas Type III collagen mRNA showed significant increase in the pericyte group at days 3, 6 and 9 compared with the fibroblast and no-cell groups. Trichrome staining revealed thick unorganized collagen fibrils in the fibroblast-treated wounds, whereas pericyte-treated wounds contained thinner and more alligned collagen fibrils. Tumor necrosis factor (TNF)-α mRNA levels were increased in the fibroblast-treated wounds compared with pericyte-treated wounds. Discussion The addition of pericytes may confer beneficial effects to wound healing resulting in reduced recruitment of inflammatory cells and collagen I deposition, potential to enhance wound closure and better collagen alignment promoting stronger tissue.

Lamin B1 is a component of the nuclear lamina and plays a critical role in maintaining nuclear architecture, regulating gene expression and modulating chromatin positioning. We have previously shown that LMNB1 gene duplications cause autosomal dominant leukodystrophy (ADLD), a fatal adult onset demyelinating disease. The mechanisms by which increased LMNB1 levels cause ADLD are unclear. To address this, we used a transgenic mouse model where Lamin B1 overexpression is targeted to oligodendrocytes. These mice showed severe vacuolar degeneration of the spinal cord white matter together with marked astrogliosis, microglial infiltration, and secondary axonal damage. Oligodendrocytes in the transgenic mice revealed alterations in histone modifications favoring a transcriptionally repressed state. Chromatin changes were accompanied by reduced expression of genes involved in lipid synthesis pathways, many of which are known to play important roles in myelin regulation and are preferentially expressed in oligodendrocytes. Decreased lipogenic gene expression resulted in a significant reduction in multiple classes of lipids involved in myelin formation. Many of these gene expression changes and lipid alterations were observed even before the onset of the phenotype, suggesting a causal role. Our findings establish, for the first time, a link between LMNB1 and lipid synthesis in oligodendrocytes, and provide a mechanistic framework to explain the age dependence and white matter involvement of the disease phenotype. These results have implications for disease pathogenesis and may also shed light on the regulation of lipid synthesis pathways in myelin maintenance and turnover. SIGNIFICANCE STATEMENT Autosomal dominant leukodystrophy (ADLD) is fatal neurological disorder caused by increased levels of the nuclear protein, Lamin B1. The disease is characterized by an age-dependent loss of myelin, the fatty sheath that covers nerve fibers. We have studied a mouse model where Lamin B1 level are increased in oligodendrocytes, the cell type that produces myelin in the CNS. We demonstrate that destruction of myelin in the spinal cord is responsible for the degenerative phenotype in our mouse model. We show that this degeneration is mediated by reduced expression of lipid synthesis genes and the subsequent reduction in myelin enriched lipids. These findings provide a mechanistic framework to explain the age dependence and tissue specificity of the ADLD disease phenotype.

Cisplatin induces nausea and emesis, even with antiemetic supportive care. To assess platinum exposure, which could activate nausea and emesis, we quantitated platinum in the brain and various organs, and hindbrain and spinal cord substance P, a key neuropeptide for the neuronal signaling of nausea and emesis.Musk shrews, a model species for nausea and emesis research, were dosed intraperitoneally with 20 mg/kg cisplatin and euthanized at up to 72 h after injection. Concentrations of platinum were quantitated in plasma ultrafiltrate, plasma, lung, kidney, combined forebrain and midbrain, hindbrain, and spinal cord by flameless atomic absorption spectrometry. Hindbrains and spinal cords were analyzed for substance P by immunohistochemistry after injection of 20 or 30 mg/kg.Plasma ultrafilterable platinum concentrations decreased rapidly till 60 min after dosing and then more slowly by 24 h. The concentrations of total platinum in both the fore- and midbrain and the hindbrain were similar at all time points and were at least 20-fold lower than plasma total platinum concentrations. There were no significant changes in substance P immunoreactivity after cisplatin dosing. Histology revealed damage to the renal cortex by 72 h after injection of cisplatin.This is the first study to examine platinum concentrations in musk shrews after administration of cisplatin and delineate substance P immunohistochemical staining in the hindbrain and spinal cord of this species. The platinum concentrations detected in the brain could potentially contribute to the neurological side effects of cisplatin, such as nausea and emesis.

Elongation factor, RNA polymerase II, 2 (ELL2) is an RNA Pol II elongation factor with functional properties similar to ELL that can interact with the prostate tumor suppressor EAF2. In the prostate, ELL2 is an androgen response gene that is upregulated in benign prostatic hyperplasia (BPH). We recently showed that ELL2 loss could enhance prostate cancer cell proliferation and migration, and that ELL2 gene expression was downregulated in high Gleason score prostate cancer specimens. Here, prostate-specific deletion of ELL2 in a mouse model revealed a potential role for ELL2 as a prostate tumor suppressor in vivo. Ell2-knockout mice exhibited prostatic defects including increased epithelial proliferation, vascularity and PIN lesions similar to the previously determined prostate phenotype in Eaf2-knockout mice. Microarray analysis of prostates from Ell2-knockout and wild-type mice on a C57BL/6J background at age 3 months and qPCR validation at 17 months of age revealed a number of differentially expressed genes associated with proliferation, cellular motility and epithelial and neural differentiation. OncoPrint analysis identified combined downregulation or deletion in prostate adenocarcinoma cases from the Cancer Genome Atlas (TCGA) data portal. These results suggest that ELL2 and its pathway genes likely play an important role in the development and progression of prostate cancer.

Introduction: The SOD1G93A mouse model of ALS, demonstrates hind limb paralysis beginning at 90 - 100 days of age with stage 4 paralysis at 125 days of age and progressive neuromuscular loss. Materials and Methods: To determine whether deficiency of functional SOD1 influenced parameters of hematopoiesis, long-term bone marrow cultures were established from ALS and control mice. Bone marrow stromal cell lines derived from LTBMCs were tested for clonogenic radiation survival. We tested the effect of bone marrow transplant after total body irradiation on delay of paralysis. Results: SOD1G93A marrow cultures demonstrated significant increase in production of hematopoietic progenitor cells (p < 0.0001) and overall longevity of production of hematopoietic cells (p = 0.0354), and bone marrow stromal cell lines were significantly radioresistant (D0 = 1.33 ± 0.09, and ñ = 8.57 ± 1.8) compared to control C57BL/6J mice (D0 = 1.59 ± 0.11, p = 0.117; and ñ = 3.4 ± 0.4, p= 0.0466). Total body irradiation and bone marrow transplantation with GFP+ donor marrow demonstrated a significant increase in paralysis free interval from 129.2 ± 3.0 to 240.7 ± 21.1 days (p = 0.0010), normalization of blood/brain barrier permeability, and increase in M2 marrow origin microglial cells in proximity to degenerating anterior horn cell/motor neurons. Isolated brain and spinal cord irradiation did not prolong the paralysis free interval (129.0 ± 2.7 days, p = 0.7748). Conclusions: The results of hematopoiesis in LTBMCs in the absence of functional SOD1 showed improved LTBMC longevity and radioresistance of marrow stromal cells both unexpected pleiotropic effects of the SOD1G93A genotype. Marrow transplant after TBI prolonged the paralysis free interval in ALS mice. Citation Format: Andrew Henderson, Michael W. Epperly, Renee Fisher, Donna Shields, Lora Rigatti, Christopher Donnelly, Simon Watkins, Joel S. Greenberger. Increased longevity of continuous bone marrow cultures and radioresistance of bone marrow stromal cells from SOD193A ALS (Amyotrophic Lateral Sclerosis) mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5155.

Introduction: The SOD1G93A mouse model of ALS, demonstrates hind limb paralysis beginning at 90 - 100 days of age with stage 4 paralysis at 125 days of age and progressive neuromuscular loss. Materials & Methods: To determine whether deficiency of functional SOD1 influenced parameters of hematopoiesis, long-term bone marrow cultures were established from ALS and control mice. Bone marrow stromal cell lines derived from LTBMCs were tested for clonogenic radiation survival. We tested the effect of bone marrow transplant after total body irradiation on delay of paralysis. Results: SOD1G93A marrow cultures demonstrated significant increase in production of hematopoietic progenitor cells (p < 0.0001) and overall longevity of production of hematopoietic cells (p = 0.0354), and bone marrow stromal cell lines were significantly radioresistant (D0 = 1.33 ± 0.09, and ñ = 8.57 ± 1.8) compared to control C57BL/6J mice (D0 = 1.59 ± 0.11, p = 0.117; and ñ = 3.4 ± 0.4, p= 0.0466). Total body irradiation and bone marrow transplantation with GFP+ donor marrow demonstrated a significant increase in paralysis free interval from 129.2 ± 3.0 to 240.7 ± 21.1 days (p = 0.0010), normalization of blood/brain barrier permeability, and increase in M2 marrow origin microglial cells in proximity to degenerating anterior horn cell/motor neurons. Isolated brain and spinal cord irradiation did not prolong the paralysis free interval (129.0 ± 2.7 days, p = 0.7748). Conclusions: The results showing increased longevity of hematopoiesis in LTBMCs of marrow from mice displaying an absence of SOD1 and the radioresistance of derived bone marrow stromal cell lines represent two unexpected pleiotrophic effects of the SOD1 G93A genotype. Further studies will be required to determine how marrow transplant after TBI prolonged the paralysis free interval in these ALS mice. Disclosures No relevant conflicts of interest to declare.

Background: Collagenous colitis is a chronic inflammatory bowel disease of unknown etiology. It is fairly common in adult humans, but rare in infants, and has been associated with autoimmune disorders. Methods: We report four infant baboons (age 7–12 months) that had received a transplant at 3 months of age and subsequent immunosuppressive therapy for periods of 4–10 months. All presented identical symptoms within a period of 4 weeks, including weight loss associated with chronic watery diarrhea that was unresponsive to standard antimicrobial treatment. Results: Clinical chemistry evaluations were within normal ranges, viral causes were ruled out, and fecal and blood cultures were repeatedly negative. At necropsy, two infant baboons were found to have a form of collagenous colitis. In the remaining two baboons that had identical clinical features, immunosuppressive therapy was discontinued and treatment with budesonide was initiated. Both baboons recovered and remained well on no medication until the end of follow-up (24 months). Conclusions: Collagenous colitis has occasionally been reported in patients with organ transplants. It has been reported only once previously in baboons. The four cases reported here strongly suggest that 1) clinical features as well as histopathological findings of collagenous colitis in baboons are very similar to those in human patients; 2) it was associated with the immunocompromised state of the baboons, as two nonimmunosuppressed age-matched baboons in close proximity did not develop the condition; and 3) it may have had an infectious origin, as all four cases developed within a 4-week period of time. (Inflamm Bowel Dis 2012;)

A major hurdle in transplantation (Tx) is the long waiting time to obtain a donor organ. This problem is particularly striking in infants with congenital heart defects, who often require heart Tx at a young age (1). The pig could provide an alternative source of organs if the immunologic barriers could be overcome (2). The initial barrier is related to the presence of natural (preformed) antibodies (Abs) in the recipient directed to antigens on the vascular endothelium of the pig organ (3, 4). Ab binding initiates activation of the complement cascade, resulting in hyperacute rejection (5, 6). Natural immunoglobulin (Ig)-M Abs develop during infancy, a process believed to be associated with colonization of the gastrointestinal tract with bacteria/viruses that express galactose-α1,3-galactose (Gal) antigens (7–9). This natural Ab production is considered to be T-cell independent (10), although there is some evidence that it may be T-cell dependent (11). The development of anti-Gal Abs is similar to that of other Abs directed to carbohydrate antigens, for example, the A and B blood group antigens (12). Natural anti-A/B Abs are usually absent during the first 3 months of life in humans and baboons but subsequently develop during the first year (9, 13). The relative absence of Abs during the first few months has provided a “window of opportunity” during which AB-incompatible (AB-I) organ Tx can be carried out successfully (14). Infants that received an AB-I organ did not reject the graft, and subsequently developed donor-specific B-cell tolerance, defined by the absence of donor-specific Abs in the presence of a graft and normal development of nonspecific Abs, which was confirmed by a negative antidonor agglutination titer and enzyme-linked immunosorbent spot (ELISPOT) (15, 16). Anti-pig Abs are primarily directed against Gal antigens expressed on pig cells, which share their core structure with the ABO antigens (12). Similar to anti-A/B Abs, Abs to wild-type (WT) pig cells in human and baboon infants do not develop until approximately 3 months of age (9). Early studies demonstrated that hyperacute rejection does not occur after WT pig heart Tx into untreated newborn baboons (17). We hypothesized that if an infant received a pig organ graft before the development of natural anti-pig Abs, these Abs might never develop, and B-cell tolerance to the pig graft would result. We have investigated this by carrying out WT pig or AB-I baboon artery patch Tx in baboons of 3 months of age. We could not confirm that this hypothesis is correct; however, all baboons that received anti-CD154mAb-based immunosuppressive therapy (IS), irrespective of the presence or source of a graft, showed inhibited development of both anti-pig and anti-A/B Abs compared with their age-matched controls, suggesting that natural Abs are T-cell dependent.

Background Dupuytren’s disease (DD) is a slow, progressive fibroproliferative disorder affecting the palms of the hands. The disease is characterized by the formation of collagen rich- cords which gradually shorten by the action of myofibroblasts resulting in finger contractures. It is a disease that is confined to humans, and a major limiting factor in investigating this disorder has been the lack of a faithful animal model that can recapitulate its distinct biology. The aim of this study was to develop such a model by determining if Dupuytren’s disease (DD)- and control carpal tunnel (CT)-derived fibroblasts could survive in the forepaw of the nude rats and continue to exhibit the distinct characteristics they display in in vitro cultures. Methods 1x107 fluorescently labeled DD- and CT-derived fibroblasts were transplanted into the left and right forepaws of nude rats respectively. Cells were tracked at regular intervals for a period of two months by quantifying emitted fluorescent signal using an IVIS imaging system. After a period of 62 days rat forepaw connective tissues were harvested for histology and total RNA was isolated. Human-specific probes were used to perform real time RT-PCR assays to examine the expression patterns of gene products associated with fibrosis in DD. Rat forepaw skin was also harvested to serve as an internal control. Results Both CT- and DD-derived fibroblasts survived for a period of 62 days, but DD-derived cells showed a significantly greater level of persistent fluorescent signal at the end of this time than did CT-derived cells. mRNA expression levels of α-smooth muscle actin (α-SMA), type I- and type III- collagens were all significantly elevated in the forepaw receiving DD cord-derived fibroblasts in comparison to CT-derived fibroblasts. Masson’s trichrome stain confirmed increased collagen deposition in the forepaw that was injected with DD cord-derived fibroblasts. Conclusions For the first time we describe an animal model for Dupuytren’s disease at the orthotopic anatomical location. We further show that gene expression differences between control (CT) and diseased (DD) derived fibroblasts persist when these cells are transplanted to the forepaw of the nude rat. These preliminary findings indicate that, with further refinements, this animal model holds promise as a baseline for investigating novel therapeutic regimens to determine an effective strategy in treating DD.

Benzaldehyde dimethane sulfonate (DMS612, NSC281612, BEN) is an alkylator with activity against renal cell carcinoma, currently in phase I trials. In blood, BEN is rapidly metabolized into its highly reactive carboxylic acid (BA), presumably the predominant alkylating species. We hypothesized that BEN is metabolized to BA by aldehyde dehydrogenase (ALDH) and aimed to increase BEN exposure in blood and tissues by inhibiting ALDH with disulfiram, thereby shifting BA production from blood to tissues. Female CD2F1 mice were dosed with 20 mg/kg BEN iv alone or 24 h after 300 mg/kg disulfiram ip. BEN, BA, and metabolites were quantitated in plasma and urine, and toxicities were assessed. BEN had a plasma t½

Multiple genetic alterations are associated with prostate carcinogenesis. Tumor-suppressor genes phosphatase and tensin homolog deleted on chromosome 10 (Pten) and androgen upregulated gene 19 (U19), which encodes ELL-associated factor 2 (EAF2), are frequently inactivated or downregulated in advanced prostate cancers. Previous studies showed that EAF2 knockout caused tumors in multiple organs and prostatic intraepithelial neoplasia (PIN) in mice. However, EAF2-knockout mice did not develop prostate cancer even at 2 years of age. To further define the roles of EAF2 in prostate carcinogenesis, we crossed the Pten +/- and EAF2 +/- mice in the C57/BL6 background to generate EAF2 -/- Pten +/-, Pten +/-, EAF2 -/- and wild-type mice. The prostates from virgin male mice with the above four genotypes were analyzed at 7 weeks, 19 weeks and 12 months of age. Concomitant loss of EAF2 function and inactivation of one Pten allele induced spontaneous prostate cancer in 33% of the mice. Prostatic tissues from intact EAF2 -/- Pten +/- mice exhibited higher levels of phospho-Akt, -p44/42 and microvessel density. Moreover, phospho-Akt remained high after castration. Consistently, there was a synergistic increase in prostate epithelial proliferation in both intact and castrated EAF2 -/- Pten +/- mice. Using laser-capture microdissection coupled with real-time reverse transcription-PCR, we confirmed that co-downregulation of EAF2 and Pten occurred in > 50% clinical prostate cancer specimens with Gleason scores of 8-9 (n = 11), which is associated with poor prognosis. The above findings together demonstrated synergistic functional interactions and clinical relevance of concurrent EAF2 and Pten downregulation in prostate carcinogenesis.

Background: Large animals treated with immunosuppressive drugs for preclinical experiments of transplantation have increased risks of infection, which can be compounded by the induction of diabetes if islet transplantation is planned. Methods: We report our experience with severe sepsis in two young cynomolgus monkeys and five pigs that were subjected to diabetes induction, immunosuppressive therapy, or islet allotransplantation. Results: In two monkeys and five pigs, infection was associated with a syndrome of profound hypoglycemia accompanied by severe acidosis, which was resistant to treatment. We do not believe that this syndrome has been reported previously by others. Conclusions: Despite treatment, this syndrome complicated the interpretation of blood glucose readings as a measure of islet graft function and resulted in death or the need for euthanasia in all seven animals. We tentatively suggest that the syndrome may be related to the presence of microorganisms that metabolize glucose and produce lactate.

Purpose: A practical radiation mitigator must be safe in pregnant females. Irradiation during pregnancy can induce fetal death, stunt growth, and/or lead to teratogenic and carcinogenic effects dependent on stage of gestation. We evaluated the mitochondrial targeted GS-nitroxide mitigator JP4-039 for effects on total body irradiated (TBI) pregnant mice. Methods: Timed pregnant C57Bl/6NHsd mice at E13 were irradiated to 3 Gy, subgroups were injected IV 24 hr later (E14) with JP4-039 in F14 emulsion (4 ug JP4-039 in 100 ul of F14). Mice were followed for number of pups born, weight of pups at day 5 after birth, and for number of survivors at time of weaning. In other studies of irradiation effects, new- born pups, on the day of delivery, were euthanized, fixed, sectioned and examined microscopically. Results: Nonirradiated mice showed 97 ± 2% of newborn surviving until weaning. Pups born to 3 Gy irradiated E13 pregnant mice had decreased survival (8.3 ± 8.7%) (p < 0.0001). Control nonirradiated pregnant mice receiving JP4-039/F14 or F14 alone showed no effect on pup survival (85 ± 10% at weaning) (p = 0.1453). The survival of pups from 3 Gy irradiated E13 pregnant mice that received JP4-039/F14 24 hr after irradiation was significantly decreased (45 ± 16.4% compared to nonirradiated controls (p = 0.0230). All newborn pups were weighed at 5 days after birth: those surviving 3 Gy in utero had significantly decreased weight of 1.64 ± 0.04 g compared to 2.73 ± 0.08 g for nonirradiated controls (p < 0.0001). In contrast, while the 3 Gy TBI E13 irradiated pups from mothers that received JP4-039/F14 showed no significant weight change compared to control nonirradiated pups (2.44 ± 0.15 and 2.73 ± 0.08, respectively, p = 0.0799). Their weight was significantly increased compared to the 3 Gy irradiated group (2.44 ± 0.15 and 1.64 ± 0.04 g, respectively, p = 0.0433). Pups from nonirradiated mothers that were administered JP4-039/F14 had a significantly increased weight on day 5 compared to nonirradiated mice (3.32 ± 0.10 and 2.73 ± 0.08 g, respectively, p < 0.0001). Microscopic examination of irradiated pups dying at day of birth, revealed: 1) increased number of hematopoietic precursors in liver and decreased glycogen stores in the hepatocytes; 2)adrenal glands were enlarged and contain severely hypertrophied cortical cells; 3) brain, showed necrosis and loss of parenchyma within the intermediate zone of white matter and cell debris in the lateral ventricles. These changes were not observed in pups from JP4-039/F14 treated mothers, sacrificed on the day of birth. Conclusions: Treatment of total body irradiated E13 pregnant mice at E14 with JP4-039/F14 was safe and effective as a radiation mitigator, led to increased numbers of surviving newborns, improved growth and development, and after weaning over 21 days, and increased body weight with no late deaths. Supported by NIAID/NIH U19-AI068021 Citation Format: Michael W. Epperly, Lora Rigatti, Tracy Dixon, Song Li, Joel S. Greenberger. JP4-039/F14 treatment of E13 pregnant mice 24 hours after total body irradiation (TBI) improves survival, growth and development of fetal mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1660.

Introduction: Transgenic FVB/n mice with the HPV E7 oncogene under the control of the CK14 promoter were bred with Fancd2+/- (129/Sv background) mice to obtain K14E7 Fancd2-/- mice Park, et al. Cancer Research, 70(23): 9959, 2010), in which delivery of 4-nitroquinoline N-oxide in drinking water produces oral and esophageal adenomas. The hematopoietic system, of these mice was studied, using long term bone marrow cultures (LTBMCs) from Fancd2-/- (SV129), K14E7 transgenic (FVB/n), and K14E7 Fancd2-/- mice. Methods: LTBMCs were established by flushing bone marrow into T-25 flasks from which bone marrow stromal cell lines and IL3-dependent hematopoietic cell lines were developed. The cell lines were analyzed using irradiation survival curves, Western analysis for protein expression. K14E7 Fancd2-/- IL3 dependent clonal cell lines of the IL3-dependent cell lines were established by flow cytometry. Clonal lines were expanded and tested for tumorigenicity by injecting 1 × 106 cells of each of four clones into flanks of K14E7 Fancd2+/+ mice. Results: K14E7 Fancd2-/- LTBMCs were similar to those from Sv/129 Fancd2-/-: 1) hematopoiesis was shortened when compared to K14E7 Fancd2+/+ or Fancd2+/+ LTBMCs, respectively. Stromal cell line irradiation survival curves with K14E7 Fancd2+/+ and Fancd2-/- cell lines showed (Do = 1.48 ± 0.05 and 1.52 ± 0.15 Gy) they were radiosensitive compared to those from K14E7 Fancd2+/+ or Sv/129 Fancd2+/+ cells (Do = 2.33 ± 0.11 and 2.23 ± 0.01 Gy, p = 0.0043 and 0.0368, respectively). Fancd2-/- stromal cells were more radiosensitive than Fancd2+/+ cells (decreased shoulder on the survival curve of n = 1.5 ± 0.5 compared to 4.7 ± 0.3 (p = 0.0126). K14E7Fancd2+/+ cells had an n of 1.9 ± 0.3 compared to 3.5 ± 0.1 (p = 0.0309) for Sv/129 cells. In contrast, hematopoietic IL-3 dependent K14E7Fancd2+/+ and Sv/129 Fancd2+/+ cell lines were similar (Do = 2.15 ± 0.4 and 1.92 ± 0.06, p = 0.6469, respectively). Confirming prior data, (Berhane et. al, Rad Res 182: 35, 2014) Sv/129 Fancd2-/- IL-3 dependent cell lines were radioresistant compared to Fancd2+/+ cell lines (Do = 2.12 ± 0.12 and 1.92 ± 0.06, respectively, p = 0.0236). In contrast, K14E7 Fancd2-/- cell lines were radiosensitive compared to K14E7 Fancd2+/+ cell lines (1.44 ± 0.13 and 2.15 ± 0.28, respectively, p = 0.0498). Hematopoietic and marrow stromal cell lines from K14E7Fancd2-/- marrow expressed cytokeratin 14 and E7 oncogene by Western analysis. K14E7 Fancd2-/- (but no other genotype derived) IL-3 dependent cell lines transformed in vitro to dense tumor cells lines. Cloned sublines were injected into K14E7 Fancd2+/- mice producing plasmacytomas. Conclusions: Expression of the E7 oncogene in the K14/E7 Fancd2-/- mouse marrow derived IL-3 dependent cell lines leads to radiosensitivity and tumorgenicity. Supported by NIAID/NIH U19-A 1068021 and the Fanconi Anemia Research Foundation. Citation Format: Joel S. Greenberger, Lora Rigatti, Aranee Sivanathan, Shaonan Cao, Xichen Zhang, Donna Shields, Darcy Franicola, Michael Epperly. Expression of the HPV E7 oncogene in K14E7Fancd2-/- mouse long term bone marrow culture derived hematopoietic cells produces malignant plasmacytomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1655.

The liver changes with age, leading to an impaired ability to respond to hepatic insults and increased incidence of liver disease in the elderly. Therefore, there is critical need for rapid model systems to study aging-related liver changes. One potential opportunity is murine models of human progerias or diseases of accelerated aging. Ercc1(-/Δ) mice model a rare human progeroid syndrome caused by inherited defects in DNA repair. To determine whether hepatic changes that occur with normal aging occur prematurely in Ercc1(-/Δ) mice, we systematically compared liver from 5-month-old progeroid Ercc1(-/Δ) mice to old (24-36-month-old) wild-type (WT) mice. Both displayed areas of necrosis, foci of hepatocellular degeneration, and acute inflammation. Loss of hepatic architecture, fibrosis, steatosis, pseudocapillarization, and anisokaryosis were more dramatic in Ercc1(-/Δ) mice than in old WT mice. Liver enzymes were significantly elevated in serum of Ercc1(-/Δ) mice and old WT mice, whereas albumin was reduced, demonstrating liver damage and dysfunction. The regenerative capacity of Ercc1(-/Δ) liver after partial hepatectomy was significantly reduced. There was evidence of increased oxidative damage in Ercc1(-/Δ) and old WT liver, including lipofuscin, lipid hydroperoxides and acrolein, as well as increased hepatocellular senescence. There was a highly significant correlation in genome-wide transcriptional changes between old WT and 16-week-old, but not 5-week-old, Ercc1(-/Δ) mice, emphasizing that the Ercc1(-/Δ) mice acquire an aging profile in early adulthood.There are strong functional, regulatory, and histopathological parallels between accelerated aging driven by a DNA repair defect and normal aging. This supports a role for DNA damage in driving aging and validates a murine model for rapidly testing hypotheses about causes and treatment for aging-related hepatic changes.