TNF-α modulates genome-wide redistribution of ΔNp63α/TAp73 and NF-κB cREL interactive binding on TP53 and AP-1 motifs to promote an oncogenic gene program in squamous cancer.

The Cancer Genome Atlas (TCGA) network study of 12 cancer types (PanCancer 12) revealed frequent mutation of TP53, and amplification and expression of related TP63 isoform ΔNp63 in squamous cancers. Further, aberrant expression of inflammatory genes and TP53/p63/p73 targets were detected in the PanCancer 12 project, reminiscent of gene programs comodulated by cREL/ΔNp63/TAp73 transcription factors we uncovered in head and neck squamous cell carcinomas (HNSCCs). However, how inflammatory gene signatures and cREL/p63/p73 targets are comodulated genome wide is unclear. Here, we examined how the inflammatory factor tumor necrosis factor-α (TNF-α) broadly modulates redistribution of cREL with ΔNp63α/TAp73 complexes and signatures genome wide in the HNSCC model UM-SCC46 using chromatin immunoprecipitation sequencing (ChIP-seq). TNF-α enhanced genome-wide co-occupancy of cREL with ΔNp63α on TP53/p63 sites, while unexpectedly promoting redistribution of TAp73 from TP53 to activator protein-1 (AP-1) sites. cREL, ΔNp63α and TAp73 binding and oligomerization on NF-κB-, TP53- or AP-1-specific sequences were independently validated by ChIP-qPCR (quantitative PCR), oligonucleotide-binding assays and analytical ultracentrifugation. Function of the binding activity was confirmed using TP53-, AP-1- and NF-κB-specific REs or p21, SERPINE1 and IL-6 promoter luciferase reporter activities. Concurrently, TNF-α regulated a broad gene network with cobinding activities for cREL, ΔNp63α and TAp73 observed upon array profiling and reverse transcription-PCR. Overlapping target gene signatures were observed in squamous cancer subsets and in inflamed skin of transgenic mice overexpressing ΔNp63α. Furthermore, multiple target genes identified in this study were linked to TP63 and TP73 activity and increased gene expression in large squamous cancer samples from PanCancer 12 TCGA by CircleMap. PARADIGM inferred pathway analysis revealed the network connection of TP63 and NF-κB complexes through an AP-1 hub, further supporting our findings. Thus, inflammatory cytokine TNF-α mediates genome-wide redistribution of the cREL/p63/p73, and AP-1 interactome, to diminish TAp73 tumor suppressor function and reciprocally activate NF-κB and AP-1 gene programs implicated in malignancy.

A mutant p53/let-7i-axis-regulated gene network drives cell migration, invasion and metastasis.

Most p53 mutations in human cancers are missense mutations resulting in a full-length mutant p53 protein. Besides losing tumor suppressor activity, some hotspot p53 mutants gain oncogenic functions. This effect is mediated in part, through gene expression changes due to inhibition of p63 and p73 by mutant p53 at their target gene promoters. Here, we report that the tumor suppressor microRNA let-7i is downregulated by mutant p53 in multiple cell lines expressing endogenous mutant p53. In breast cancer patients, significantly decreased let-7i levels were associated with missense mutations in p53. Chromatin immunoprecipitation and promoter luciferase assays established let-7i as a transcriptional target of mutant p53 through p63. Introduction of let-7i to mutant p53 cells significantly inhibited migration, invasion and metastasis by repressing a network of oncogenes including E2F5, LIN28B, MYC and NRAS. Our findings demonstrate that repression of let-7i expression by mutant p53 has a key role in enhancing migration, invasion and metastasis.

High frequencies of leukemia stem cells in poor-outcome childhood precursor-B acute lymphoblastic leukemias.

In order to develop a xenograft model to determine the efficacy of new therapies against primary human precursor-B acute lymphoblastic leukemia (ALL) stem cells (LSCs), we used the highly immunodeficient non-obese diabetic (NOD).Cg-Prkdc(scid)IL2rg(tmlWjl)/SzJ (NOD-severe combined immune deficient (scid) IL2rg(-/-)) mouse strain. Intravenous transplantation of 2 of 2 ALL cell lines and 9 of 14 primary ALL cases generated leukemia-like proliferations in recipient mice by 1-7 months after transplant. Leukemias were retransplantable, and the immunophenotypes, gene rearrangements and expression profiles were identical or similar to those of the original primary samples. NOD-scid mice transplanted with the same primary samples developed similar leukemias with only a slightly longer latency than did NOD-scid-IL2Rg(-/-) mice. In this highly sensitive NOD-scid-IL2Rg(-/-)-based assay, 1-100 unsorted primary human ALL cells from five of five tested patients, four of whom eventually experienced leukemia relapse, generated leukemias in recipient mice. This very high frequency of LSCs suggests that a hierarchical LSC model is not valuable for poor-outcome ALL.

Epigenomic alterations and gene expression profiles in respiratory epithelia exposed to cigarette smoke condensate.

Limited information is available regarding epigenomic events mediating initiation and progression of tobacco-induced lung cancers. In this study, we established an in vitro system to examine epigenomic effects of cigarette smoke in respiratory epithelia. Normal human small airway epithelial cells and cdk-4/hTERT-immortalized human bronchial epithelial cells (HBEC) were cultured in normal media with or without cigarette smoke condensate (CSC) for up to 9 months under potentially relevant exposure conditions. Western blot analysis showed that CSC mediated dose- and time-dependent diminution of H4K16Ac and H4K20Me3, while increasing relative levels of H3K27Me3; these histone alterations coincided with decreased DNA methyltransferase 1 (DNMT1) and increased DNMT3b expression. Pyrosequencing and quantitative RT-PCR experiments revealed time-dependent hypomethylation of D4Z4, NBL2, and LINE-1 repetitive DNA sequences; up-regulation of H19, IGF2, MAGE-A1, and MAGE-A3; activation of Wnt signaling; and hypermethylation of tumor suppressor genes such as RASSF1A and RAR-beta, which are frequently silenced in human lung cancers. Array-based DNA methylation profiling identified additional novel DNA methylation targets in soft-agar clones derived from CSC-exposed HBEC; a CSC gene expression signature was also identified in these cells. Progressive genomic hypomethylation and locoregional DNA hypermethylation induced by CSC coincided with a dramatic increase in soft-agar clonogenicity. Collectively, these data indicate that cigarette smoke induces 'cancer-associated' epigenomic alterations in cultured respiratory epithelia. This in vitro model may prove useful for delineating early epigenetic mechanisms regulating gene expression during pulmonary carcinogenesis.

Aeromonas species associated with necrotizing enteritis and septicemia in an adult male ostrich (Struthio camelus).

A deceased 10-yr-old male ostrich was diagnosed with severe necrotizing enteritis and septicemia. The bird was inappetent for 3 wk and had neurologic signs 2 days prior to death. Macroscopically, no significant lesions were noted aside from congestion of the liver, kidneys, and spleen. Histopathology revealed severe fibrinonecrotic enteritis,associated with large numbers of gram-negative bacteria, multifocal fibrinoid necrosis in portal arteries, accumulation of fibrin in hepatic sinusoids, myocardial degeneration, and necrosis. There was also squamous metaplasia in the glands of the esophagus and external ears. A gram-negative rod was isolated in pure culture from intestine, liver, lungs, and trachea and identified as an Aeromonas species. The concentration of vitamin A in the liver was extremely low. The lesions seen in the intestine and liver and the isolation of an Aeromonas sp. from various tissues strongly suggest that this bacterium was the cause of the necrotizing enteritis, septicemia, and death of this ostrich. Vitamin A deficiency might have predisposed the bird to the Aeromonas infection.

Genetic diversity of bovine ulcerative mammary dermatitis-associated Treponema.

The etiology of bovine ulcerative mammary dermatitis (UMD) is poorly characterized. The goal of this study was to genetically analyze spirochetes present in UMD lesions. DNA prepared from UMD lesion biopsies and from spirochetes cultured from the corresponding lesion biopsies was PCR amplified using primers for the 16S rDNA-tRNA(ile) intergenic spacer region (ISR) of Treponema 16S-23S rDNA. Analysis of cloned ISR amplicons from three cultivable UMD-associated spirochetes indicated that two isolates cluster closely with cultivable papillomatous digital dermatitis (PDD)-associated and human-associated Treponema phylotypes, while the remaining isolate is unique. Analysis of ISR amplicons from UMD lesion biopsies identified additional not-yet-cultivable Treponema phylotypes. Our results revealed the presence of a genetically diverse Treponema population in an UMD lesion.

Cutaneous pythiosis in a nestling white-faced ibis.

A nestling white-faced ibis (Plegadis chihi) with multifocal skin ulcerations on the wings, neck, head, and limbs was found in a wetland agricultural region of the central valley in California. Pathologic, microbiologic, and molecular findings were consistent with restricted, cutaneous infection by the oomycete Pythium insidiosum. The microscopic features of the disease, including intense, necrotizing eosinophilic and granulomatous inflammation, are similar to those previously described in mammals. Pythiosis, which is most typical in tropical and subtropical climates, has recently emerged in California as a cause of cutaneous and enteric disease in horses and dogs, respectively. Environmental stability and persistence of a "water-mold" in the arid central valley of California could be associated with agricultural and community watering practices. To the best of the authors' knowledge, this is the first published report of pythiosis in birds.

Listeriosis in a cockatiel (Nymphicus hollandicus).

Listeriosis was diagnosed in a 4-yr-old female cockatiel (Nymphicus hollandicus) that died after exhibiting clinical signs that included a fluffed-up appearance, weakness, and loss of weight of several days duration. Grossly, the bird was moderately emaciated, and the liver and spleen were enlarged. Microscopically, there was mild-to-moderate inflammation associated with rod-shaped, gram-positive bacteria in the liver, spleen, kidneys, adrenal glands, bone marrow, and esophagus. Listeria monocytogenes was isolated from the liver, trachea, and intestine. The isolate was identified as type 1 by agglutination with specific antisera, and it further identified as belonging to serovar group 1/2a, 3a by multiplex polymerase chain reaction assay. Listeria monocytogenes also was detected in affected tissues by immunohistochemistry.

Molecular markers expressed in cultured and freshly isolated interstitial cells of Cajal.

Located within the tunica muscularis of the gastrointestinal (GI) tract are networks of cells known as interstitial cells of Cajal (ICC). ICC are critical for important basic functions of GI motility such as generation and propagation of slow-wave pacemaker activity and reception of regulatory inputs from the enteric nervous system. We have developed a novel procedure to identify and isolate individual ICC from freshly dispersed cell preparations of the murine small intestine and gastric fundus and to determine differential transcriptional expression We have compared the expression profiles of pacemaker ICC isolated from the murine small intestine (IC-MY) and ICC involved in neurotransmission from the gastric fundus (IC-IM). We have also compared expression profiles between ICC and smooth muscle cells (SMC) and between freshly isolated ICC and cultured ICC. Cultured ICC express smooth muscle myosin, whereas freshly dispersed ICC do not. All cell types express muscarinic receptor types M(2) and M(3), neurokinin receptors NK(1) and NK(3), and inhibitory receptor VIP-1, whereas only cultured ICC and SMC express VIP-2. Both cultured and freshly dispersed IC-IM and IC-MY express the soluble form of stem cell factor, whereas SMC from the gastric fundus express only the membrane-bound form.

Structure and promoter analysis of the human unc-33-like phosphoprotein gene. E-box required for maximal expression in neuroblastoma and myoblasts.

The human unc-33-like phosphoprotein (hUlip/CRMP-4) is a member of a family of developmentally regulated genes that are highly expressed in the nervous system. Mutations in the C. elegans unc-33 gene lead to worms with abnormal movements. The hUlip gene encodes a 570-amino acid protein with 98% homology to its murine (Ulip) (Byk, T., Dobransky, T., Cifuentes-Diaz, C., and Sobel, A. (1996) J. Neurosci. 16, 688-701) and rat (CRMP-4) (Wang, L. H., and Strittmatter, S. M. (1996) J. Neurosci. 16, 6197-6207) counterparts (Gaetano, C., Matsuo, T., and Thiele, C. J. (1997) J. Biol. Chem. 272, 12195-12201). The hUlip gene was isolated from a human genomic library. It contains 15 exons, including an exon defined by an anaplastic oligodendroglioma expressed sequence tag, and spans at least 61.7 kilobases. hUlip lacks sequences corresponding to the first six exons found in unc-33. unc-33 exons correspond to homologous hUlip exons as follows: VII to 1 and 2, VIII to 3-9, IX to 10-12, and X to 13 and 14. Using the hUlip clone 1 phage, fluorescence in situ hybridization analysis indicates that the hybridization signal localizes to human chromosome 5q32. Deletion analysis of 5'-flanking sequences delineated the sequences sufficient to express a reporter gene in both neuroblastoma cells and myoblasts. A consensus MyoD/myogenin binding site is located in a region of the downstream promoter that is nearly identical to its mouse homologue. Mutagenesis shows that this conserved MyoD/myogenin site is necessary for full promoter activity in both myoblasts and neuroblastoma cells.

Farm- and host-level risk factors for papillomatous digital dermatitis in Chilean dairy cattle.

A cross-sectional study was conducted in southern Chile between January and March, 1996, to identify risk factors for papillomatous digital dermatitis (PDD) in lactating dairy and dual-purpose cows. A total of 3,265 cows from 22 farms were examined in the milking parlor for PDD lesions. Additional information was collected from dairies' computerized records and by direct interview of managers. Data were analyzed using logistic and logistic-binomial regression (with dairy as a random-effect term). German Red-Pied (dual-purpose) cows were significantly (P < 0.05) less likely (odds ratio (OR) = 0.3) to have PDD lesions than German Black-Pied and Holstein crossbreds. First-parity cows had the highest odds of PDD, and odds diminished, in a dose-effect manner, as parity increased. Odds of PDD increased with increasing days in lactation. Cows that calved during winter were more likely to have PDD (OR = 1.4) than those calving at any other season. Cows on farms that bought heifers in the past 10 years had a 3-fold increase in the odds of PDD compared to those on farms that never bought heifers. Loose-housed cows had a higher risk of PDD (OR = 7), followed by cows in free stalls or in open corrals (OR = 2.8 and 1.3, respectively), compared to cows on pasture all year. Cows on dairies that used a footbath during 1996 were less likely (OR = 0.3) to have PDD than those in dairies not using one. Parlor type was associated with PDD, but this was likely an effect of parlor design on ease of inspection of cows' feet. A policy of trimming all cows' vs. only lame cows' feet and a policy about buying adult cows did not have significant effects on PDD risk.

Specific chromosomal aberrations and amplification of the AIB1 nuclear receptor coactivator gene in pancreatic carcinomas.

To screen pancreatic carcinomas for chromosomal aberrations we have applied molecular cytogenetic techniques, including fluorescent in situ hybridization, comparative genomic hybridization, and spectral karyotyping to a series of nine established cell lines. Comparative genomic hybridization revealed recurring chromosomal gains on chromosome arms 3q, 5p, 7p, 8q, 12p, and 20q. Chromosome losses were mapped to chromosome arms 8p, 9p, 17p, 18q, 19p, and chromosome 21. The comparison with comparative genomic hybridization data from primary pancreatic tumors indicates that a specific pattern of chromosomal copy number changes is maintained in cell culture. Metaphase chromosomes from six cell lines were analyzed by spectral karyotyping, a technique that allows one to visualize all chromosomes simultaneously in different colors. Spectral karyotyping identified multiple chromosomal rearrangements, the majority of which were unbalanced. No recurring reciprocal translocation was detected. Cytogenetic aberrations were confirmed using fluorescent in situ hybridization with probes for the MDR gene and the tumor suppressor genes p16 and DCC. Copy number increases on chromosome 20q were validated with a probe specific for the nuclear receptor coactivator AIB1 that maps to chromosome 20q12. Amplification of this gene was identified in six of nine pancreatic cancer cell lines and correlated with increased expression.

In breast cancer, amplification of the steroid receptor coactivator gene AIB1 is correlated with estrogen and progesterone receptor positivity.

The AIB1 gene was isolated upon microdissection of the homogeneously staining regions observed in breast cancer cell lines. It was subsequently shown to map at a region at 20q12 that is frequently amplified in breast tumors. In a screen of breast tumor cell lines, of all the genes mapping to the region, AIB1 appeared to be the most consistently amplified and overexpressed. AIB1 shares homology with the SRC-1 family of nuclear receptor coactivators. It was found to interact in a ligand-dependent manner with the estrogen receptor (ER) and to result in increased levels of estrogen-dependent transcription. These properties could be of important biological significance in breast and ovarian cancerigenesis, and we were, therefore, interested in determining whether the amplification of the AIB1 gene was associated with a particular phenotype or subgroup in these tumors. We tested a population of 1157 breast and 122 ovarian tumors in which DNA amplification had been determined previously at 15 chromosomal locations. Amplification of the AIB1 gene was observed in 4.8% of breast cancers and 7.4% of ovarian cancers. In breast tumors, AIB1 was correlated with ER and progesterone receptor positivity, as well as with tumor size. Correlation was also observed with the amplification of MDM2 and FGFR1 genes, but interestingly, no correlation was found with the amplification of CCND1, which is known to be strongly associated with ER. Furthermore, analyzing at 20q12-q13 range, we show the existence of three amplification cores, represented by AIB3/AIB4, AIB1, and RMC20C001. AIB1 and CCND1 amplifications may, thus, represent two different subsets of ER-positive breast tumors.

AIB1, a steroid receptor coactivator amplified in breast and ovarian cancer.

Members of the recently recognized SRC-1 family of transcriptional coactivators interact with steroid hormone receptors to enhance ligand-dependent transcription. AIB1, a member of the SRC-1 family, was cloned during a search on the long arm of chromosome 20 for genes whose expression and copy number were elevated in human breast cancers. AIB1 amplification and overexpression were observed in four of five estrogen receptor-positive breast and ovarian cancer cell lines. Subsequent evaluation of 105 unselected specimens of primary breast cancer found AIB1 amplification in approximately 10 percent and high expression in 64 percent of the primary tumors analyzed. AIB1 protein interacted with estrogen receptors in a ligand-dependent fashion, and transfection of AIB1 resulted in enhancement of estrogen-dependent transcription. These observations identify AIB1 as a nuclear receptor coactivator whose altered expression may contribute to development of steroid-dependent cancers.

Spirochetes isolated from dairy cattle with papillomatous digital dermatitis and interdigital dermatitis.

Two groups of spirochetes were isolated from papillomatous digital dermatitis (PDD) lesions in dairy cattle. The two groups could be readily differentiated on the basis of morphologic and immunologic characteristics and enzymatic activity. A spirochete isolated from an interdigital dermatitis (IDD) lesion appeared morphologically and antigenically similar to spirochetes in one of the PDD groups and exhibited an identical enzyme activity pattern. The two groups of PDD spirochetes had characteristics most consistent with the genus Treponema. The PDD and IDD isolates differed morphologically from previously described bovine Treponema spp. Although spirochetes have been observed to be one of the predominant bacterial morphotypes in PDD and IDD and are found invading the stratum spinosum and dermal papillae in PDD lesions, the significance of these spirochetes in the etiopathogenesis of PDD and IDD is presently unknown.

Diagnosis of favus (avian dermatophytosis) in Oriental breed chickens.

Chickens of various Oriental breeds (Shamo and Aseel) and crossbreeds in California's Central Valley were observed to have an unusual skin condition and feather loss. The appearance of white plaques on the comb, face, and/or ear lobes was followed by feather loss starting at the caudal base of the comb and progressing down the neck. Although the cocks were affected first, the condition spread to the hens paired with those cocks. The birds showed no other signs of illness. The affected areas were scraped and biopsied. The samples were examined histologically and by culturing on Sabouraud's dextrose agar and dermatophyte test medium. Microsporum gallinae, the causative agent of favus (avian dermatophytosis), was identified by the histological and mycological tests.

Comparison of an antimicrobial adhesive drape and povidone-iodine preoperative skin preparation in dogs.

The antimicrobial efficacy of an adhesive drape applied after a 1-minute alcohol scrub was compared to a povidone-iodine (PI) skin preparation technique in dogs. Each technique was applied to both sides of 15 adult anesthetized dogs on premeasured, clipped areas of skin. Skin bacteria were quantified before, immediately after, and 1 hour after skin preparation. Predominant skin bacteria were isolated by swabbing the skin. The percentages of bacterial reduction immediately after and 1 hour after skin preparation, percentages of negative culture results, cultures with more than five colony-forming units, and the frequency of skin reactions were calculated and analyzed statistically. Drape adhesion was assessed subjectively. The percentage reduction in skin bacteria was significant for both techniques and comparable to that reported in humans. The adhesive drape was significantly less effective in both the immediate and 1-hour periods. Lift occurred in 66% of drape applications but was not associated with high bacterial counts. Acute contact dermatitis was more frequent after skin preparation with PI. There was no difference between the techniques in recovery of potential skin pathogens. The authors conclude that application of this antimicrobial adhesive drape after a 1-minute alcohol scrub is not as effective in the reduction of skin bacteria in dogs as is PI preparation of the skin.

Comparison of three skin preparation techniques in the dog. Part 1: Experimental trial.

Premeasured, clipped areas of skin on both sides of 30 adult dogs were prepared with povidone-iodine (PI), chlorhexidine gluconate (CG) with a saline rinse, or 4% CG with a 70% isopropyl alcohol rinse. Skin bacteria were quantified with Replicating Organism Detection and Counting (RODAC) plates and cultured for identification before, immediately after, and 1 hour after skin preparation. The percentages of bacterial reduction immediately and at hour 1 and the percentages of negative cultures, cultures with more than five colony-forming units (CFUs), and skin reactions were analyzed by analysis of variance and chi-square. The percentage of reduction in skin bacteria for all techniques was significant and comparable with that reported in humans. There were no significant differences between PI and CG results except that acute contact dermatitis was observed more frequently after skin preparation with PI. The authors conclude that for similar application times, PI and 4% CG rinsed with saline or 70% isopropyl alcohol are equally effective for up to 1 hour in the preoperative skin preparation of dogs.

Comparison of three skin preparation techniques. Part 2: Clinical trial in 100 dogs.

The skin of 100 dogs undergoing clean or clean-contaminated surgical procedures was prepared with povidone-iodine (PI) or 4% chlorhexidine gluconate (CG) with saline or 70% isopropyl alcohol rinse. Skin bacteria at the incision site were quantified with Replication Organism Detection and Counting (RODAC) plates immediately before and after skin preparation in the preparation room, in the operating room, and postoperatively. The percentage of bacterial reduction, negative cultures, cultures with more than five colony-forming units, and skin reactions for each technique were calculated for each sample period and analyzed with the analysis of variance and Fischer tests. The percentage of bacterial reduction for all techniques was significant and comparable with results of a previous experimental study. There were no significant differences in percentages of bacterial reduction between PI and the CG techniques for surgical times up to 8 hours. There were fewer negative cultures and more cultures with high bacterial counts with PI than with CG and saline after the cleansing scrub. There were fewer negative cultures after surgery with CG and alcohol than with the other two techniques. Duration of the surgical procedure did not significantly affect the culture results. Significantly more skin reactions occurred with PI. The authors conclude that PI and 4% CG with a saline rinse are equally effective in antimicrobial efficacy under clinical conditions. However, 4% CG with a 70% isopropyl alcohol rinse may be inferior in residual antimicrobial activity.