TAp63 suppresses mammary tumorigenesis through regulation of the Hippo pathway.

Mechanisms regulating the transition of mammary epithelial cells (MECs) to mammary stem cells (MaSCs) and to tumor-initiating cells (TICs) have not been entirely elucidated. The p53 family member, p63, is critical for mammary gland development and contains transactivation domain isoforms, which have tumor-suppressive activities, and the ΔN isoforms, which act as oncogenes. In the clinic, p63 is often used as a diagnostic marker, and further analysis of the function of TAp63 in the mammary gland is critical for improved diagnosis and patient care. Loss of TAp63 in mice leads to the formation of aggressive metastatic mammary adenocarcinoma at 9-16 months of age. Here we show that TAp63 is crucial for the transition of mammary cancer cells to TICs. When TAp63 is lost, MECs express embryonic and MaSC signatures and activate the Hippo pathway. These data indicate a crucial role for TAp63 in mammary TICs and provide a mechanism for its role as a tumor- and metastasis-suppressor in breast cancer.

Pastoralism in the drylands of Latin America: Argentina, Chile, Mexico and Peru. / Pastoralismo en zonas áridas de Latinoamérica: Argentina, Chile, México y Perú.

This article discusses various aspects of pastoralism in the Latin American countries with the largest dryland areas. The topics covered include: social, economic and institutional issues; grasslands and their carrying capacity; production systems and productivity rates; competition for forage resources between domestic livestock and wildlife; and the health status of livestock and wildlife. Most grasslands exhibit some degree of degradation. The percentage of offspring reaching weaning age is low: 47-66% of calves and 40-80% of lambs. Some pastoralists adopt patterns of transhumance. In the main, pastoralists experience a high poverty rate and have poor access to social services. For many pastoralists, wildlife is a source of food and by-products. Argentina, Chile, Mexico and Peru have animal health control agencies, are members of the World Organisation for Animal Health (OIE) and have signed the United Nations Convention to Combat Desertification. Pastoral systems subsist mainly on income unrelated to pastoral farming. The OIE recognises all four countries as free from infection with peste des petits ruminants virus, and from rinderpest and African horse sickness. It is difficult to predict the future of pastoralism in Latin America because the situation differs from country to country. For instance, pastoralism is more important in Peru than in Argentina, where it is a more marginal activity. In the future, lack of promotion and protection policies could lead to a decline in pastoralism or to an adverse environmental impact on drylands.

Les auteurs abordent les particularités du pastoralisme dans les quatre pays d'Amérique latine dotés des plus vastes étendues de terres arides du souscontinent ; ils examinent notamment les aspects sociaux, économiques et institutionnels du pastoralisme, les pâtures et leur capacité de charge, les systèmes de production et les indices de productivité, la concurrence entre le bétail et les animaux sauvages pour le prélèvement de fourrage et le statut sanitaire respectif des animaux d'élevage et de la faune sauvage. Les prairies dédiées au pastoralisme présentent dans leur majorité un certain degré de dégradation. Le taux de survie au sevrage fluctue entre 47 % et 66 % pour les veaux et entre 40 % et 80 % pour les agneaux. Certains pasteurs adoptent des schémas de transhumance. Les populations pastorales se caractérisent par un niveau élevé de pauvreté et un accès très limité aux services sociaux. Pour de nombreux pasteurs, la faune sauvage constitue une ressource alimentaire directe mais elle fournit aussi des produits dérivés. En Argentine, au Chili, au Mexique et au Pérou, des services gouvernementaux sont chargés du contrôle de la santé animale. Ces pays sont Membres de l'Organisation mondiale de la santé animale (OIE) et signataires de la Convention des Nations Unies sur la lutte contre la désertification. Les systèmes pastoraux subsistent essentiellement grâce à des revenus autres que ceux issus de la production. Ces territoires ont été reconnus par l'OIE comme étant indemnes d'infection par les virus de la peste des petits ruminants, de la peste bovine et de la peste équine. Il est difficile de prédire l'avenir du pastoralisme en Amérique latine, en raison de la diversité des situations rencontrées d'un pays à l'autre. Par exemple, l'activité pastorale est plus importante au Pérou qu'en Argentine où elle a un caractère marginal. L'absence de politiques de promotion et de protection spécifiques pourrait se traduire à l'avenir par un déclin du pastoralisme ou par un impact écologique négatif pour ces zones arides.

Se abordan matices del pastoralismo relativos a los países con mayor extensión de zonas áridas de Latinoamérica, concretamente, los aspectos sociales, económicos e institucionales, los pastizales y su receptividad, los sistemas de producción e índices de productividad, la competencia entre ganado doméstico y fauna silvestre por el recurso forrajero, y el estatus sanitario del ganado y de los animales silvestres. La mayor parte de los pastizales presenta algún grado de deterioro. El porcentaje de crías que llega al destete fluctúa entre el 47% y el 66% en bovinos y entre el 40% y el 80% en ovinos. Algunos pastoralistas adoptan patrones de trashumancia. Los pastores se caracterizan básicamente por un índice alto de pobreza y un escaso acceso a los servicios sociales. La fauna es un recurso alimentario y una fuente de subproductos para numerosos pastores. Argentina, Chile, México y Perú cuentan con organismos destinados al control de la sanidad animal, son miembros de la OIE y han suscrito la Convención de las Naciones Unidas de Lucha contra la Desertificación. Los sistemas pastoriles subsisten principalmente a partir de ingresos ajenos a su producción. La OIE reconoce a estos territorios como libres de infección por peste de los pequeños rumiantes, por peste bovina y por peste equina. Es difícil predecir el futuro del pastoralismo en Latinoamérica debido a las diferentes situaciones de cada país. Así, por ejemplo, en Perú, esta práctica tiene mayor importancia que en Argentina, donde es más marginal. La carencia de políticas de promoción y protección podría conducir en el futuro a una disminución de la actividad o a un impacto ecológico negativo en las zonas áridas.

Genetic characterization of two gain-of-function alleles of the effector caspase DrICE in Drosophila.

Caspases are the executioners of apoptosis. Although much is known about their physiological roles and structures, detailed analyses of missense mutations of caspases are lacking. As mutations within caspases are identified in various human diseases, the study of caspase mutants will help to elucidate how caspases interact with other components of the apoptosis pathway and how they may contribute to disease. DrICE is the major effector caspase in Drosophila required for developmental and stress-induced cell death. Here, we report the isolation and characterization of six de novo drICE mutants, all of which carry point mutations affecting amino acids conserved among caspases in various species. These six mutants behave as recessive loss-of-function mutants in a homozygous condition. Surprisingly, however, two of the newly isolated drICE alleles are gain-of-function mutants in a heterozygous condition, although they are loss-of-function mutants homozygously. Interestingly, they only behave as gain-of-function mutants in the presence of an apoptotic signal. These two alleles carry missense mutations affecting conserved amino acids in close proximity to the catalytic cysteine residue. This is the first time that viable gain-of-function alleles of caspases are described in any intact organism and provides a significant exception to the expectation that mutations of conserved amino acids always abolish the pro-apoptotic activity of caspases. We discuss models about how these mutations cause the gain-of-function character of these alleles.

Functions of TAp63 and p53 in restraining the development of metastatic cancer.

Many tumours harbour mutations in the p53 tumour-suppressor gene that result in the expression of a mutant p53 protein. This mutant p53 protein has, in most cases, lost wild-type transcriptional activity and can also acquire novel functions in promoting invasion and metastasis. One of the mechanisms underlying these novel functions involves the ability of the mutant p53 to interfere with other transcription factors, including the p53 family protein TAp63. To investigate whether simultaneous depletion of both p53 and TAp63 can recapitulate the effect of mutant p53 expression in vivo, we used a mouse model of pancreatic cancer in which the expression of mutant p53 resulted in the rapid appearance of primary tumours and metastases. As shown previously, loss of one allele of wild-type (WT) p53 accelerated tumour development. A change of one WT p53 allele into mutant p53 did not further accelerate tumour development, but did promote the formation of metastasis. By contrast, loss of TAp63 did not significantly accelerate tumour development or metastasis. However, simultaneous depletion of p53 and TAp63 led to both rapid tumour development and metastatic potential, although the incidence of metastases remained lower than that seen in mutant p53-expressing tumours. TAp63/p53-null cells derived from these mice also showed an enhanced ability to scatter and invade in tissue culture as was observed in mutant p53 cells. These data suggest that depletion of TAp63 in a p53-null tumour can promote metastasis and recapitulate-to some extent-the consequences of mutant p53 expression.

Interaction between the TP63 and SHH pathways is an important determinant of epidermal homeostasis.

Deregulation of the hedgehog (HH) pathway results in overexpression of the GLI target BCL2 and is an initiating event in specific tumor types including basal cell carcinoma of the skin. Regulation of the HH pathway during keratinocyte differentiation is not well understood. We measured HH pathway activity in response to differentiation stimuli in keratinocytes. An upregulation of suppressor of fused (SUFU), a negative regulator of the HH pathway, lowered HH pathway activity and was accompanied by loss of BCL2 expression associated with keratinocyte differentiation. We used in vitro and in vivo models to demonstrate that ΔNp63α, a crucial regulator of epidermal development, activates SUFU transcription in keratinocytes. Increasing SUFU protein levels inhibited GLI-mediated gene activation in suprabasal keratinocytes and promoted differentiation. Loss of SUFU expression caused deregulation of keratinocyte differentiation and BCL2 overexpression. Using in vivo murine models, we also provide evidence of GLI-mediated regulation of the TP63 pathway. p63 expression appears essential to establish an optimally functioning HH pathway. These observations present a regulatory mechanism by which SUFU acts as an interacting node between the HH and TP63 pathways to mediate differentiation and maintain epidermal homeostasis. Disruption of this regulatory node can be an important contributor to multistep carcinogenesis.

Centrosome abnormalities and genomic instability by episomal expression of human papillomavirus type 16 in raft cultures of human keratinocytes.

Primary human keratinocytes with ectopic expression of high-risk human papillomavirus (HPV) E6 and E7 oncoproteins display abnormal centrosome numbers, multipolar mitoses, and aneusomy. However, it has not been explored whether these abnormalities can occur in cells containing HPV episomes where E6 and E7 expression is under viral transcriptional control. Here, we demonstrate that centrosome abnormalities and genomic instability occur in organotypic raft cultures of human keratinocytes with episomal HPV-16 even at low copy numbers. We conclude that HPV-16 DNA, when maintained as an episome, can disturb centrosome homeostasis and subvert genomic integrity of the host cell during early stages of the viral infection.

Role for the p53 homologue p73 in E2F-1-induced apoptosis.

The transcription factor E2F-1 induces both cell-cycle progression and, in certain settings, apoptosis. E2F-1 uses both p53-dependent and p53-independent pathways to kill cells. The p53-dependent pathway involves the induction by E2F-1 of the human tumour-suppressor protein p14ARF, which neutralizes HDM2 (human homologue of MDM2) and thereby stabilizes the p53 protein. Here we show that E2F-1 induces the transcription of the p53 homologue p73. Disruption of p73 function inhibited E2F-1-induced apoptosis in p53-defective tumour cells and in p53-/- mouse embryo fibroblasts. We conclude that activation of p73 provides a means for E2F-1 to induce death in the absence of p53.

The human papillomavirus type 16 E7 oncogene is required for the productive stage of the viral life cycle.

The production of the human papillomavirus type 16 (HPV-16) is intimately tied to the differentiation of the host epithelium that it infects. Infection occurs in the basal layer of the epithelium at a site of wounding, where the virus utilizes the host DNA replication machinery to establish itself as a low-copy-number episome. The productive stage of the HPV-16 life cycle occurs in the postmitotic suprabasal layers of the epithelium, where the virus amplifies its DNA to high copy number, synthesizes the capsid proteins (L1 and L2), encapsidates the HPV-16 genome, and releases virion particles as the upper layer of the epithelium is shed. Papillomaviruses are hypothesized to possess a mechanism to overcome the block in DNA synthesis that occurs in the differentiated epithelial cells, and the HPV-16 E7 oncoprotein has been suggested to play a role in this process. To determine whether E7 plays a role in the HPV-16 life cycle, an E7-deficient HPV-16 genome was created by inserting a translational termination linker (TTL) in the E7 gene of the full HPV-16 genome. This DNA was transfected into an immortalized human foreskin keratinocyte cell line shown previously to support the HPV-16 life cycle, and stable cell lines were obtained that harbored the E7-deficient HPV-16 genome episomally, the state of the genome found in normal infections. By culturing these cells under conditions which promote the differentiation of epithelial cells, we found E7 to be necessary for the productive stage of the HPV-16 life cycle. HPV-16 lacking E7 failed to amplify its DNA and expressed reduced amounts of the capsid protein L1, which is required for virus production. E7 appears to create a favorable environment for HPV-16 DNA synthesis by perturbing the keratinocyte differentiation program and inducing the host DNA replication machinery. These data demonstrate that E7 plays an essential role in the papillomavirus life cycle.

Establishment of the human papillomavirus type 16 (HPV-16) life cycle in an immortalized human foreskin keratinocyte cell line.

The study of human papillomaviruses (HPVs) in cell culture has been hindered because of the difficulty in recreating the three-dimensional structure of the epithelium on which the virus depends to complete its life cycle. Additionally, the study of genetic mutations in the HPV genome and its effects on the viral life cycle are difficult using the current method of transfecting molecularly cloned HPV genomes into early-passage human foreskin keratinocytes (HFKs) because of the limited life span of these cells. Unless the HPV genome transfected into the early-passage HFK extends the life span of the cell, analysis of stable transfectants becomes difficult. In this study, we have used BC-1-Ep/SL cells, an immortalized human foreskin keratinocyte cell line, to recreate the HPV-16 life cycle. This cell line exhibits many characteristics of the early-passage HFKs including the ability to stratify and terminally differentiate in an organotypic raft culture system. Because of their similarity to early-passage HFKs, these cells were tested for their ability to support the HPV-16 life cycle. The BC-1-Ep/SL cells could stably maintain two HPV genotypes, HPV-16 and HPV-31b, episomally. Additionally, when the BC-1-Ep/SL cell line was stably transfected with HPV-16 and cultured using the organotypic raft culture system (rafts), it sustained the HPV-16 life cycle. Evidence for the productive stage of the HPV-16 life cycle was provided by: DNA in situ hybridization demonstrating HPV-16 DNA amplification in the suprabasal layers of the rafts, immunohistochemical staining for L1 showing the presence of capsid protein in the suprabasal layers of the rafts, and electron microscopy indicating the presence of virus like particles (VLPs) in nuclei from cells in the differentiated layers of the rafts.

Evidence for a switch in the mode of human papillomavirus type 16 DNA replication during the viral life cycle.

The study of human papillomavirus type 16 (HPV-16) replication has been impaired because of the lack of a cell culture system that stably maintains viral replication. Recently, cervical epithelial cell populations that stably maintain HPV-16 replicons at a copy number of approximately 1,000 per cell were derived from an HPV-16-infected patient (W12 cell clone 20863 [W12-E cells]). We used neutral/neutral and neutral/alkaline two-dimensional gel electrophoretic techniques to characterize HPV-16 DNA replication in these cells. When W12-E cells were maintained in an undifferentiated state mimicking the nonproductive stage of the life cycle, HPV-16 DNA was found to replicate primarily by theta structures in a bidirectional manner. The initiation site of HPV-16 DNA replication was mapped to approximately nucleotide 100, and the termination site was mapped to between nucleotides 3398 and 5990. To study the productive stage of HPV-16 DNA replication, W12-E cells were grown under culture conditions that promote differentiation of epithelial cell types. Under these conditions, where virus-like particles were detected, the mode of viral DNA replication changed from theta structure to what is apparently a rolling circle mode. Additionally, CIN 612-9E cells, which were derived from an HPV-31-infected patient and harbor HPV-31 extrachromosomally, exhibited the same switch in the mode of DNA replication upon induction of differentiation. These data argue that a fundamental switch in the mechanism of viral DNA replication occurs during the life cycle of the papillomavirus.

Effects of different heat treatments during processing of soybean meal on nursery and growing pig performance.

Four soybean meals (SBM) were manufactured in a commercial solvent-extraction plant to give a much wider range in heat treatment than is usually found among commercially available SBM. The SBM were designated in ascending order of heat treatment as Under, Normal, Over and Rumen Escape. The nutritive value of the four meals was evaluated in a series of five feeding trials using 458 pigs: two performance and two diet preference trials with pigs weaned at 4 wk of age and one performance trial with growing pigs (17.4 kg initial weight were conducted). In both nursery and grower trials, there were no differences (P greater than .10) in performance of pigs fed the four meals. However, in the nursery trials, the severely heated meal (Rumen Escape) supported slightly lower gains (6.4%) and less desirable feed efficiency (3.5%) than the average of the other three meals. Growing pig performance was essentially the same for all meals. This suggests that older pigs either used the Rumen Escape meal more effectively than nursery pigs, or the Rumen Escape diet contained adequate digestible lysine for 17.4-kg pigs to grow optimally. In the preference studies, pigs selected between Normal- and Rumen Escape-supplemented diets. Pigs consumed 63 and 62% of the Normal diet in preference trials 1 and 2, but these differences were not significant (P greater than .10) due to the large variation among pens. These data suggest that the range of heat treatment normally found among commercially available SBM (Under, Normal and Over meals) has no effect on the nutritive value of the meal for swine.(ABSTRACT TRUNCATED AT 250 WORDS)