LRIG1 opposes epithelial-to-mesenchymal transition and inhibits invasion of basal-like breast cancer cells.

LRIG1 (leucine-rich repeat and immunoglobulin-like domain containing), a member of the LRIG family of transmembrane leucine-rich repeat-containing proteins, is a negative regulator of receptor tyrosine kinase signaling and a tumor suppressor. LRIG1 expression is broadly decreased in human cancer and in breast cancer and low expression of LRIG1 has been linked to decreased relapse-free survival. Recently, low expression of LRIG1 was revealed to be an independent risk factor for breast cancer metastasis and death. These findings suggest that LRIG1 may oppose breast cancer cell motility and invasion, cellular processes that are fundamental to metastasis. However, very little is known of LRIG1 function in this regard. In this study, we demonstrate that LRIG1 is downregulated during epithelial-to-mesenchymal transition (EMT) of human mammary epithelial cells, suggesting that LRIG1 expression may represent a barrier to EMT. Indeed, depletion of endogenous LRIG1 in human mammary epithelial cells expands the stem cell population, augments mammosphere formation and accelerates EMT. Conversely, expression of LRIG1 in highly invasive Basal B breast cancer cells provokes a mesenchymal-to-epithelial transition accompanied by a dramatic suppression of tumorsphere formation and a striking loss of invasive growth in three-dimensional culture. LRIG1 expression perturbs multiple signaling pathways and represses markers and effectors of the mesenchymal state. Furthermore, LRIG1 expression in MDA-MB-231 breast cancer cells significantly slows their growth as tumors, providing the first in vivo evidence that LRIG1 functions as a growth suppressor in breast cancer.

Who's behind that mask and cape? The Asian leopard cat's Agouti (ASIP) allele likely affects coat colour phenotype in the Bengal cat breed.

Coat colours and patterns are highly variable in cats and are determined mainly by several genes with Mendelian inheritance. A 2-bp deletion in agouti signalling protein (ASIP) is associated with melanism in domestic cats. Bengal cats are hybrids between domestic cats and Asian leopard cats (Prionailurus bengalensis), and the charcoal coat colouration/pattern in Bengals presents as a possible incomplete melanism. The complete coding region of ASIP was directly sequenced in Asian leopard, domestic and Bengal cats. Twenty-seven variants were identified between domestic and leopard cats and were investigated in Bengals and Savannahs, a hybrid with servals (Leptailurus serval). The leopard cat ASIP haplotype was distinguished from domestic cat by four synonymous and four non-synonymous exonic SNPs, as well as 19 intronic variants, including a 42-bp deletion in intron 4. Fifty-six of 64 reported charcoal cats were compound heterozygotes at ASIP, with leopard cat agouti (A(P) (be) ) and domestic cat non-agouti (a) haplotypes. Twenty-four Bengals had an additional unique haplotype (A2) for exon 2 that was not identified in leopard cats, servals or jungle cats (Felis chaus). The compound heterozygote state suggests the leopard cat allele, in combination with the recessive non-agouti allele, influences Bengal markings, producing a darker, yet not completely melanistic coat. This is the first validation of a leopard cat allele segregating in the Bengal breed and likely affecting their overall pelage phenotype. Genetic testing services need to be aware of the possible segregation of wild felid alleles in all assays performed on hybrid cats.

A conserved segmental duplication within ELA.

The assembled genomic sequence of the horse major histocompatibility complex (MHC) (equine lymphocyte antigen, ELA) is very similar to the homologous human HLA, with the notable exception of a large segmental duplication at the boundary of ELA class I and class III that is absent in HLA. The segmental duplication consists of a ∼ 710 kb region of at least 11 repeated blocks: 10 blocks each contain an MHC class I-like sequence and the helicase domain portion of a BAT1-like sequence, and the remaining unit contains the full-length BAT1 gene. Similar genomic features were found in other Perissodactyls, indicating an ancient origin, which is consistent with phylogenetic analyses. Reverse-transcriptase PCR (RT-PCR) of mRNA from peripheral white blood cells of healthy and chronically or acutely infected horses detected transcription from predicted open reading frames in several of the duplicated blocks. This duplication is not present in the sequenced MHCs of most other mammals, although a similar feature at the same relative position is present in the feline MHC (FLA). Striking sequence conservation throughout Perissodactyl evolution is consistent with a functional role for at least some of the genes included within this segmental duplication.

The strength of single crystal copper under uniaxial shock compression at 100 GPa.

In situ x-ray diffraction has been used to measure the shear strain (and thus strength) of single crystal copper shocked to 100 GPa pressures at strain rates over two orders of magnitude higher than those achieved previously. For shocks in the [001] direction there is a significant associated shear strain, while shocks in the [111] direction give negligible shear strain. We infer, using molecular dynamics simulations and VISAR (standing for 'velocity interferometer system for any reflector') measurements, that the strength of the material increases dramatically (to approximately 1 GPa) for these extreme strain rates.

Ex vivo inhibition of NF-kappaB signaling in alloreactive T-cells prevents graft-versus-host disease.

The ex vivo induction of alloantigen-specific hyporesponsiveness by costimulatory pathway blockade or exposure to immunoregulatory cytokines has been shown to inhibit proliferation, IL-2 production, and the graft-versus-host disease (GVHD) capacity of adoptively transferred T-cells. We hypothesized that inhibition of the intracellular NF-kappaB pathway in alloreactive T-cells, which is critical for T-cell activation events including IL-2 transcription, could lead to alloantigen hyporesponsiveness and loss of GVHD capacity. We demonstrate that treatment of mixed lymphocyte reaction (MLR) cultures with PS1145, a potent inhibitor of NF-kappaB activation, can induce T-cell hyporesponsiveness to alloantigen in primary and secondary responses while preserving in vitro responses to potent mitogenic stimulation. GVHD lethality in recipients of ex vivo PS1145-treated cells was profoundly inhibited. Parking of control or PS1145-treated MLR cells in syngeneic Rag(-/-) recipients resulted in intact contact hypersensitivity (CHS) responses. However, GVHD lethality capacity also was restored, suggesting that lymphopenic expansion uncoupled alloantigen hyporesponsiveness. These results indicate that the NF-kappaB pathway is a critical regulator of alloresponses and provide a novel small molecule inhibitor based approach that is effective in preventing early posttransplant GVHD lethality but that also permits donor T-cell responses to recover after a period of lymphopenic expansion.

Sensitization of human breast cancer cells to natural killer cell-mediated cytotoxicity by proteasome inhibition.

The proteasome inhibitor, bortezomib, has direct anti-tumour effects and has been demonstrated to sensitize tumour cells to tumour necrosis factor-related apoptosis-inducing ligand-mediated apoptosis. Natural killer (NK) cells are effective mediators of anti-tumour responses, both through cytotoxic granule killing and apoptosis-inducing pathways. We therefore investigated if bortezomib sensitized human breast cancer cells to killing by the human NK cell line, NK-92. Bortezomib was unable to sensitize MDA-231 breast cancer cells to NK cell-mediated killing in short-term in vitro assays. However, bortezomib did cause these cells to up-regulate apoptosis-related mRNA as well as death receptors on the cell surface. In a long-term in vitro tumour outgrowth assay that allows NK cells to use their full repertoire of killing pathways, bortezomib sensitized three breast cancer cell lines to NK cell-mediated killing, which led to greater anti-tumour effects than either treatment alone. We then used a xenogeneic mouse model in which CB-17 SCID mice were injected with human breast cancer cells. This model displayed the effectiveness of NK-92 cells, but the addition of bortezomib did not increase the survival further or reduce the number of lung metastases in tumour-bearing mice. However, while bortezomib was highly cytotoxic to NK-92 cells in vitro, bortezomib treatment in vivo did not decrease NK-92 function, suggesting that through alternative dosing or timing of bortezomib, greater efficacy may occur from combined therapy. These data demonstrate that combined treatment of human breast cancer with bortezomib and NK cells has the potential to generate superior anti-tumour responses than either therapy alone.

The feline genome.

The domestic cat has a long history of informing human biology, from early studies of comparative anatomy to the present genetic characterization of many feline genetic disease models. Nearly half of these diseases have homologous counterparts in human. Difficulties studying these defects in humans provide model organisms, like the domestic cat, with a unique opportunity to further inform human hereditary and infectious disease. Here I review the progress in the development of genomic mapping resources, the recent acquisition of a feline 2x genome sequence, and how these tools now equip feline geneticists to identify and characterize genes in cats causing comparable diseases or phenotypes in other species. The availability of such a mapping resource will enable positional cloning approaches and stimulate further development and use of the domestic cat as a model for human disease, while also enhancing the health of the species itself. The cat gene map also provides a useful tool in multispecies comparative genomic analyses to better understand the causes and consequences of chromosome breakage and evolution.

Constitutive expression of functional CD40 on mouse renal cancer cells: induction of Fas and Fas-mediated killing by CD40L.

CD40, a member of the TNF receptor superfamily, is expressed on B cells, dendritic cells, and some tumor cells, including melanoma and bladder carcinoma. In this study, we report that both mouse and human renal carcinoma cells (RCC) also constitutively express functional CD40. Treatment of mouse RCC with CD40L induced strong expression of genes and proteins for ICAM-1 and Fas, and this expression was further enhanced by combining CD40L with IFN-gamma. Similar effects were demonstrated using an agonist anti-CD40 antibody. The increased levels of Fas expression on RCC after treatment with CD40L plus IFN-gamma resulted in potent killing by either FasL-positive effector cells or agonistic anti-Fas antibody. The combination of CD40L plus IFN-gamma also significantly enhanced killing of RCC by tumor-specific CTL lines. Our results demonstrate that constitutively expressed CD40 is functionally active and may provide a molecular target for the development of new approaches to the treatment of RCC.

Phylogenetic assessment of introns and SINEs within the Y chromosome using the cat family felidae as a species tree.

The cat family Felidae was used as a species tree to assess the phylogenetic performance of genes, and their embedded SINE elements, within the nonrecombining region of the Y chromosome (NRY). Genomic segments from single-copy X-Y homologs SMCY, UBE1Y, and ZFY (3,604 bp) were amplified in 36 species of cat. These genes are located within the X-degenerate region of the NRY and are thought to be molecular "fossils" that ceased conventional recombination with the X chromosome early within the placental mammal evolution. The pattern and tempo of evolution at these three genes is significant in light of the recent, rapid evolution of the family over approximately 12 Myr and provides exceptional support for each of the eight recognized felid lineages, as well as clear diagnostic substitutions identifying nearly all species. Bootstrap support and Bayesian posterior probabilities are uniformly high for defining each of the eight monophyletic lineages. Further, the preferential use of specific target-site motifs facilitating SINE insertion is empirically supported by sequence analyses of SINEs embedded within the three genes. Target-site insertion is thought to explain the contradiction between intron phylogeny and results of the SMCY SINE phylogeny that unites distantly related species. Overall, our data suggest X-degenerate genes within the NRY are singularly powerful markers and offer a valuable patrilineal perspective in species evolution.

Mapping of 53 loci in American mink (Mustela vison).

Fifty-three genes were mapped in the American mink genome using polymerase chain reaction (PCR)-based analysis of a Chinese hamster-American mink somatic cell hybrid panel. Heterologous primers designed for cat gene mapping were used in this study. Forty-nine of these loci were localized into expected chromosome regions according to Zoo-FISH data, whereas four loci--ALPL, CDC20, ERF-2, and Fc(Mv)23617--were mapped out of expected conserved regions. PCR products amplified with primers corresponding to these four markers were partly sequenced and verified using BLAST. The results showed the homology to be more than 90% between mink and human or cat counterparts. At present, the gene map of American mink has expanded to 127 loci.

Second-generation integrated genetic linkage/radiation hybrid maps of the domestic cat (Felis catus).

We report construction of second-generation integrated genetic linkage and radiation hybrid (RH) maps in the domestic cat (Felis catus) that exhibit a high level of marker concordance and provide near-full genome coverage. A total of 864 markers, including 585 coding loci (type I markers) and 279 polymorphic microsatellite loci (type II markers), are now mapped in the cat genome. We generated the genetic linkage map utilizing a multigeneration interspecies backcross pedigree between the domestic cat and the Asian leopard cat (Prionailurus bengalensis). Eighty-one type I markers were integrated with 247 type II markers from a first-generation map to generate a map of 328 loci (320 autosomal and 8 X-linked) distributed in 47 linkage groups, with an average intermarker spacing of 8 cM. Genome coverage spans approximately 2,650 cM, allowing an estimate for the genetic length of the sex-averaged map as 3,300 cM. The 834-locus second-generation domestic cat RH map was generated from the incorporation of 579 type I and 255 type II loci. Type I markers were added using targeted selection to cover either genomic regions underrepresented in the first-generation map or to refine breakpoints in human/feline synteny. The integrated linkage and RH maps reveal approximately 110 conserved segments ordered between the human and feline genomes, and provide extensive anchored reference marker homologues that connect to the more gene dense human and mouse sequence maps, suitable for positional cloning applications.

Radiation hybrid mapping of 304 novel microsatellites in the domestic cat genome.

Effective utilization of the domestic cat as an animal model for hereditary and infectious disease requires the development and implementation of high quality gene maps incorporating microsatellites and conserved coding gene markers. Previous feline linkage and radiation hybrid maps have lacked sufficient microsatellite coverage on all chromosomes to make effective use of full genome scans. Here we report the isolation and genomic mapping of 304 novel polymorphic repeat loci in the feline genome. The new loci were mapped in the domestic cat radiation hybrid panel using an automated fluorescent TAQ-Man based assay. The addition of these 304 microsatellites brings the total number of microsatellites mapped in the feline genome to 580, and the total number of loci placed onto the RH map to 1,126. Microsatellites now span every autosome with an average spacing of roughly one polymorphic STR every five centimorgans, and full genome coverage of one marker every 2.7 megabases. These loci now provide a useful tool for undertaking full-genome scans to identify genes associated with phenotypes of interest, such as those relating to hereditary disease, coat color, patterning and morphology. These resources can also be extended to the remaining 36 species of the cat family for population genetic and evolutionary genomic analyses.

Resolution of the early placental mammal radiation using Bayesian phylogenetics.

Molecular phylogenetic studies have resolved placental mammals into four major groups, but have not established the full hierarchy of interordinal relationships, including the position of the root. The latter is critical for understanding the early biogeographic history of placentals. We investigated placental phylogeny using Bayesian and maximum-likelihood methods and a 16.4-kilobase molecular data set. Interordinal relationships are almost entirely resolved. The basal split is between Afrotheria and other placentals, at about 103 million years, and may be accounted for by the separation of South America and Africa in the Cretaceous. Crown-group Eutheria may have their most recent common ancestry in the Southern Hemisphere (Gondwana).

Effects of prolactin on hematopoiesis.

The presence of extra-pituitary prolactin and its cognitive receptors in the hematopoietic micro-environment raises the question of whether prolactin plays a role in lympho-hematopoiesis and under what conditions. Current studies suggest that endogenous prolactin does not play a significant role under normal steady-state conditions. Rather, prolactin has been implicated as a 'stress hormone', functioning to restore hematopoietic homeostasis under conditions of dysregulation. The stress response of prolactin as well as its complex relationship with other hormones and factors has resulted in conflicting reports in the literature regarding prolactin's role in lympho-hematopoiesis. A review of this literature is provided as well as discussion of conditions under which lymphohematopoietic activity of prolactin may be evident.

Immunobiology of natural killer cells and bone marrow transplantation: merging of basic and preclinical studies.

Natural killer (NK) cells mediate acute rejection of bone marrow, but not solid tissue, allografts in lethally irradiated mice. Precisely how and why this rejection occurs is still unclear. In allogeneic bone marrow transplantation (BMT), a spectrum of results is possible; one result can be marrow graft failure due to host rejection of the graft by NK and T cells and, at the opposite spectrum, the occurrence of graft-versus-host disease (GVHD). Donor NK cells, however, appear capable of improving donor engraftment without giving rise to GVHD and thus may be of use as an immunotherapy following BMT. As NK-cell inhibitory receptors play a role in bone marrow cell rejection, these same inhibitory receptors may also affect NK responses towards tumor cells. It has been demonstrated that blocking the interaction of inhibitory receptors with MHC determinants on tumor cells can result in greater antitumor effects. Thus, NK cells are capable of mediating both positive and negative effects during BMT depending on whether they are of host versus donor origin and their state of activation. Understanding their role in BMT provides insights as to their physiological roles and points the way to potential clinical uses.

The diverse role of chemokines in tumor progression: prospects for intervention (Review).

Chemokines, proteins chemotactic for leukocytes and non-leukocytes, have been intensively studied for their role in tumor growth and metastasis. Recent work has shown that particular chemokines may have multiple effects on tumors including promoting growth, angiogenesis, metastasis, and suppression of the immune response to cancer, while other chemokines inhibit tumor mediated angiogenesis and promote anti-tumor immune responses. Increasing biological evidence supports the hypothesis that tumor-generated chemokines provide more than simply angiogenic signals. Tumor-derived chemokines may potentially act as inhibitors of anti-tumor immune responses as well as autocrine growth factors for the tumor. The complexity and redundancy of tumor chemokine expression suggests that a single chemokine target for tumor therapy may not be appropriate. Indeed, multiple target therapy including blockade of tumor enhancing chemokines while delivering or inducing the secretion of anti-tumor chemokines is the approach that currently holds the most promise. The role of chemokines in tumor biology as well as various means of blocking chemokines in cancer models in order to develop successful therapeutic strategies will be discussed.

Evolution of mammalian genome organization inferred from comparative gene mapping.

Comparative genome analyses, including chromosome painting in over 40 diverse mammalian species, ordered gene maps from several representatives of different mammalian and vertebrate orders, and large-scale sequencing of the human and mouse genomes are beginning to provide insight into the rates and patterns of chromosomal evolution on a whole-genome scale, as well as into the forces that have sculpted the genomes of extant mammalian species.

Molecular dating and biogeography of the early placental mammal radiation.

The timing and phylogenetic hierarchy of early placental mammal divergences was determined based on combined DNA sequence analysis of 18 gene segments (9779 bp) from 64 species. Using rooted and unrooted phylogenies derived from distinct theoretical approaches, strong support for the divergence of four principal clades of eutherian mammals was achieved. Minimum divergence dates of the earliest nodes in the placental mammal phylogeny were estimated with a quartet-based maximum-likelihood method that accommodates rate variation among lineages using conservative fossil calibrations from nine different nodes in the eutherian tree. These minimum estimates resolve the earliest placental mammal divergence nodes at periods between 64 and 104 million years ago, in essentially every case predating the Cretaceous-Tertiary (K-T) boundary. The pattern and timing of these divergences allow a geographic interpretation of the primary branching events in eutherian history, likely originating in the southern supercontinent Gondwanaland coincident with its breakup into Africa and South America 95-105 million years ago. We propose an integrated genomic, paleontological, and biogeographic hypothesis to account for these earliest splits on the placental mammal family tree and address current discrepancies between fossil and molecular evidence.