The cell cycle inhibitor p21 controls T-cell proliferation and sex-linked lupus development.

Here we show that the cell-cycle regulator p21 is involved in immune system function. T lymphocytes from p21-/- mice exhibit significant proliferative advantage over wild-type cells following prolonged stimulation, but not after primary activation. Consistent with this, p21-deficient mice accumulate abnormal amounts of CD4+ memory cells, and develop loss of tolerance towards nuclear antigens. Similar to human lupus, female p21-deficient mice develop antibodies against dsDNA, lymphadenopathy, and glomerulonephritis, leading to decreased viability. These data demonstrate a specialized role for p21 in the control of T-cell proliferation, tolerance to nuclear antigens, and female-prone lupus. These findings could be the basis for new therapeutic approaches to lupus.

Short telomeres result in organismal hypersensitivity to ionizing radiation in mammals.

Here we show a correlation between telomere length and organismal sensitivity to ionizing radiation (IR) in mammals. In particular, fifth generation (G5) mouse telomerase RNA (mTR)(-/)- mice, with telomeres 40% shorter than in wild-type mice, are hypersensitive to cumulative doses of gamma rays. 60% of the irradiated G5 mTR(-/)- mice die of acute radiation toxicity in the gastrointestinal tract, lymphoid organs, and kidney. The affected G5 mTR(-/)- mice show higher chromosomal damage and greater apoptosis than similarly irradiated wild-type controls. Furthermore, we show that G5 mTR(-/)- mice show normal frequencies of sister chromatid exchange and normal V(D)J recombination, suggesting that short telomeres do not significantly affect the efficiency of DNA double strand break repair in mammals. The IR-sensitive phenotype of G5 mTR(-/)- mice suggests that telomere function is one of the determinants of radiation sensitivity of whole animals.

Tumorigenic activity of p21Waf1/Cip1 in thymic lymphoma.

The cell cycle inhibitor p21Waf1/Cip1 is among the most important mediators of the tumor suppressor p53. However, there is increasing evidence indicating that p21 could favor tumorigenesis in specific cell types. In particular, the absence of p21 delays the development of thymic lymphomas induced either by ataxia-telangiectasia mutated deficiency or by ionizing irradiation. Here, we extend these observations to the context of p53-deficient mice. The absence of p21 results in a significant extension of the lifespan of p53-null and p53-haploinsufficient mice, and this effect can be attributed exclusively to a decrease in the incidence of spontaneous thymic lymphomas. Specifically, despite the occurrence of a variety of tumor types in the context of p53 deficiency, the only tumors that were significantly impaired by the absence of p21 were thymic lymphomas. Moreover, the absence of p21 also delays the incidence of radiation-induced thymic lymphomas in p53-deficient mice. Interestingly, p21-deficient lymphomas have a higher apoptotic rate than p21-proficient lymphomas, and this could be on the basis of the delayed incidence of thymic lymphomas in the absence of p21. Together, our results indicate that p21 plays an oncogenic role restricted to thymic lymphomas that is mechanistically independent of p53 and associated to a lower tumor apoptotic rate.

ΔNp63α promotes adhesion of metastatic prostate cancer cells to the bone through regulation of CD82.

ΔNp63α is a critical mediator of epithelial development and stem cell function in a variety of tissues including the skin and breast, while overexpression of ΔNp63α acts as an oncogene to drive tumor formation and cancer stem cell properties in squamous cell carcinoma. However, with regards to the prostate, while ΔNp63α is expressed in the basal stem cells of the mature gland, during adenocarcinoma development, its expression is lost and its absence is used to clinically diagnose the malignant state. Surprisingly, here we identify a sub-population of bone metastatic prostate cancer cells in the PC3 cell line that express ΔNp63α. Interestingly, we discovered that ΔNp63α favors adhesion and stem-like growth of these cells in the bone microenvironment. In addition, we show that these properties require expression of the target gene CD82. Together, this work uncovers a population of bone metastatic prostate cancer cells that express ΔNp63α, and provides important information about the mechanisms of bone metastatic colonization. Finally, we identify metastasis-promoting properties for the tetraspanin family member CD82.

Tumor susceptibility of p21(Waf1/Cip1)-deficient mice.

The cell cycle regulator p21 mediates the ability of the tumor suppressor p53 to arrest cellular proliferation. We have examined the involvement of p21 in tumor suppression by following a large cohort of p21-deficient mice for an extended period of time. We report that p21-deficient mice develop spontaneous tumors at an average age of 16 months, whereas wild-type mice are tumor-free beyond 2 years of age. The tumors arising in p21-null mice derive from a variety of cell types and include hematopoietic ( approximately 65% of the tumors), endothelial ( approximately 20%), and epithelial ( approximately 10%) tumors. We have also studied radiation-induced carcinogenesis to test whether, in this setting, p53 exerts its tumor suppressor activity mainly through apoptosis, rather than through p21-mediated cell-cycle arrest. Concurring with this, p21-deficient mice did not show increased susceptibility to radiation-induced carcinogenesis. On the contrary, they were protected relative to wild-type mice. We conclude that p21, by mediating p53-dependent cell-cycle arrest, plays a significant role in tumor suppression.

PARP-2 sustains erythropoiesis in mice by limiting replicative stress in erythroid progenitors.

Erythropoiesis is a tightly regulated process in which multipotential hematopoietic stem cells produce mature red blood cells. Here we show that deletion of poly(ADP-ribose) polymerase-2 (PARP-2) in mice leads to chronic anemia at steady state, despite increased erythropoietin plasma levels, a phenomenon not observed in mice lacking PARP-1. Loss of PARP-2 causes shortened lifespan of erythrocytes and impaired differentiation of erythroid progenitors. In erythroblasts, PARP-2 deficiency triggers replicative stress, as indicated by the presence of micronuclei, the accumulation of γ-H2AX (phospho-histone H2AX) in S-phase cells and constitutive CHK1 and replication protein A phosphorylation. Transcriptome analyses revealed the activation of the p53-dependent DNA-damage response pathways in PARP-2-deficient cells, culminating in the upregulation of cell-cycle and cell death regulators, concomitant with G2/M arrest and apoptosis. Strikingly, while loss of the proapoptotic p53 target gene Puma restored hematocrit levels in the PARP-2-deficient mice, loss of the cell-cycle regulator and CDK inhibitor p21 leads to perinatal death by exacerbating impaired fetal liver erythropoiesis in PARP-2-deficient embryos. Although the anemia displayed by PARP-2-deficient mice is compatible with life, mice die rapidly when exposed to stress-induced enhanced hemolysis. Our results pinpoint an essential role for PARP-2 in erythropoiesis by limiting replicative stress that becomes essential in the absence of p21 and in the context of enhanced hemolysis, highlighting the potential effect that might arise from the design and use of PARP inhibitors that specifically inactivate PARP proteins.

Molecular bases of tropism in the PUR46 cluster of transmissible gastroenteritis coronaviruses.

Transmissible gastroenteritis coronavirus (TGEV) infects both, the enteric and the respiratory tract of swine. S protein, that is recognized by the cellular receptor, has been proposed that plays an essential role in controlling the dominant tropism. The genetic relationship of S gene from different enteric strains and non-enteropathogenic porcine respiratory coronaviruses (PRCVs) was determined. A correlation between tropism and the genetic structure of the S gene was established. PRCVs, derived from enteric isolates have a large deletion at the N-terminus of the S protein. Interestingly, two respiratory isolates, attenuated Purdue type virus (PTV-ATT) and Toyama (TOY56) have a full-length S gene. PTV-ATT has two specific amino acid differences with the S protein of the enteric viruses. One is located around position 219, within the deleted area, suggesting that alterations around this amino acid may result in the loss of enteric tropism. To study the role of different genes in tropism, a cluster of viruses closely related to PUR46 strain was analyzed. All of them have been originated by accumulating point mutations from a common, virulent isolate which infected the enteric tract. During their evolution these viruses have lost, virulence first, and then, enteric tropism. Sequencing analysis proved that enteric tropism could be lost without changes in ORFs 3a, 3b, 4, 6, and 7, and in 3'-end untranslated regions (3'-UTR). To study the role of the S protein in tropism recombinants were obtained between an enteric and a respiratory virus of this cluster. Analysis of the recombinants supported the hypothesis on the role in tropism of S protein domain around position 219.

[Prevalence of atopic dermatitis in schoolchildren from Cartagena (Spain) and relationship with sex and pollution]. / Prevalencia de la dermatitis atópica en escolares de Cartagena y su relación con el sexo y la contaminación.

BACKGROUND: Atopic dermatitis is favored by exogenous factors, such as air pollution interacting with a genetic predisposition. OBJECTIVE: The aim of the present study was to evaluate the influence of sex and air pollution in the city of Cartagena on the prevalence of atopic dermatitis. DESIGN AND METHODS: We performed a cross-sectional study using the ISAAC questionnaire in schoolchildren aged 13 and 14 years old from Cartagena (Murcia). The influence of sex and the schools' location in two distinct areas (polluted and unpolluted) on the prevalence of atopic dermatitis and its severity (described as being awakened by nocturnal itching) was analyzed. RESULTS: The prevalence of atopic eczema was 6.3 %. Atopic eczema was severe in 19 % of the cases. Risk factors for atopic eczema were female sex (OR 2.19 95 % CI: 1.59-3. 02) and attending school in a polluted area (OR: 1.83, 95 % CI 1.01-1.87) but these factors were not associated with greater severity. CONCLUSIONS: Air pollution is associated with a higher prevalence of atopic eczema and there is a trend that this eczema is more severe. The condition was more prevalent among girls.

[Association between atopic dermatitis, allergic rhinitis and asthma in schoolchildren aged 13-14 years old]. / Asociación entre dermatitis atópica, rinitis alérgica y asma en escolares de 13 y 14 años.

BACKGROUND: Atopic dermatitis is closely related to other atopic diseases, especially asthma and allergic rhinitis. Although there is a certain agreement that suffering from atopic eczema predisposes to asthma and allergic rhinitis, the risk of developing these diseases should be quantified in populations following a standardized method, which is the objective of this study. METHOD: Using the questionnaire from the International Study of Asthma and Allergy in Childhood (ISAAC), we performed a cross-sectional study of all schoolchildren aged 13 and 14 years old in the city of Cartagena (Murcia, Spain). The relationship between atopic dermatitis and its severity with asthma, allergic asthma, and allergic rhinitis was analyzed. RESULTS: Quantification of risk showed that schoolchildren with atopic eczema had a 3-fold risk of allergic rhinitis (OR: 3.33; 95 % CI: 2.45-4.54), a 4-fold risk of asthma (OR: 3.85; 95 % CI: 2.74-5.42) and a 5-fold risk of allergic asthma (OR: 4.91; 95 % CI: 3.17-7.59) compared with schoolchildren without atopic eczema. The severity of eczema was also directly related to that of asthma and rhinitis.

ZRF1 controls the retinoic acid pathway and regulates leukemogenic potential in acute myeloid leukemia.

Acute myeloid leukemia (AML) is frequently linked to epigenetic abnormalities and deregulation of gene transcription, which lead to aberrant cell proliferation and accumulation of undifferentiated precursors. ZRF1, a recently characterized epigenetic factor involved in transcriptional regulation, is highly overexpressed in human AML, but it is not known whether it plays a role in leukemia progression. Here, we demonstrate that ZRF1 depletion decreases cell proliferation, induces apoptosis and enhances cell differentiation in human AML cells. Treatment with retinoic acid (RA), a differentiating agent currently used to treat certain AMLs, leads to a functional switch of ZRF1 from a negative regulator to an activator of differentiation. At the molecular level, ZRF1 controls the RA-regulated gene network through its interaction with the RA receptor α (RARα) and its binding to RA target genes. Our genome-wide expression study reveals that ZRF1 regulates the transcription of nearly half of RA target genes. Consistent with our in vitro observations that ZRF1 regulates proliferation, apoptosis, and differentiation, ZRF1 depletion strongly inhibits leukemia progression in a xenograft mouse model. Finally, ZRF1 knockdown cooperates with RA treatment in leukemia suppression in vivo. Taken together, our data reveal that ZRF1 is a key transcriptional regulator in leukemia progression and suggest that ZRF1 inhibition could be a novel strategy to be explored for AML treatment.

Cooperative role between p21cip1/waf1 and p27kip1 in premature senescence in glandular proliferative lesions in mice.

Cellular senescence has been considered a novel target for cancer therapy. It has also been pointed out that p21(cip1/waf1) and p27(kip1) cyclin-dependent kinase inhibitors (CKIs) play a role in cellular senescence in some tumor types. Therefore, in order to address the possibility of a cooperative role between p21 and p27 proteins in senescence in vivo we analyzed cellular senescence in spontaneous glandular proliferative lesions (adrenal, thyroid and pituitary glands) in a double-KO mice model, using γH2AX, p53, p16, PTEN and Ki67 as senescence markers. The results obtained showed that p21p27 double-null mice had the lowest number of γH2AX positive cells in glandular hyperplasias and benign tumors. Also, in this group, Ki67 proliferation index correlated with a lower immunohistochemical expression of γH2AX and p53. The expression of p16 and PTEN do not seem to cause synergism of senescence in the benign lesions analyzed in p21p27 double-KO mice. These observations suggest an intrinsic cooperation between p21 and p27 CKIs in the activation of stress-induced cellular senescence and tumor progression in vivo, which would be a physiological mechanism to prevent tumor cell proliferation.

Snail1 controls TGF-ß responsiveness and differentiation of mesenchymal stem cells.

The Snail1 transcriptional repressor plays a key role in triggering epithelial-to-mesenchymal transition. Although Snail1 is widely expressed in early development, in adult animals it is limited to a subset of mesenchymal cells where it has a largely unknown function. Using a mouse model with inducible depletion of Snail1, here we demonstrate that Snail1 is required to maintain mesenchymal stem cells (MSCs). This effect is associated to the responsiveness to transforming growth factor (TGF)-ß1 that shows a strong Snail1 dependence. Snail1 depletion in conditional knockout adult animals causes a significant decrease in the number of bone marrow-derived MSCs. In culture, Snail1-deficient MSCs prematurely differentiate to osteoblasts or adipocytes and, in contrast to controls, are resistant to the TGF-ß1-induced differentiation block. These results demonstrate a new role for Snail1 in TGF-ß response and MSC maintenance.

Loss of poly(ADP-ribose) polymerase-2 leads to rapid development of spontaneous T-cell lymphomas in p53-deficient mice.

Poly(ADP-ribose) polymerase-2 (Parp-2) belongs to a family of enzymes that catalyse poly(ADP-ribosyl)ation of proteins. Parp-2 deficiency in mice (Parp-2(-/-)) results in reduced thymic cellularity associated with increased apoptosis in thymocytes, defining Parp-2 as an important mediator of T-cell survival during thymopoiesis. To determine whether there is a link between Parp-2 and the p53 DNA-damage-dependent apoptotic response, we have generated Parp-2/p53-double-null mutant mice. We found that p53(-/-) backgrounds completely restored the survival and development of Parp-2(-/-) thymocytes. However, Parp-2-deficient thymocytes accumulated high levels of DNA double-strand breaks (DSB), independently of the p53 status, in line with a function of Parp-2 as a caretaker promoting genomic stability during thymocytes development. Although Parp-2(-/-) mice do not have spontaneous tumours, Parp-2 deficiency accelerated spontaneous tumour development in p53-null mice, mainly T-cell lymphomas. These data suggest a synergistic interaction between Parp-2 and p53 in tumour suppression through the role of Parp-2 in DNA-damage response and genome integrity surveillance, and point to the potential importance of examining human tumours for the status of both genes.

Disease states associated with telomerase deficiency appear earlier in mice with short telomeres.

Mice deficient for the mouse telomerase RNA (mTR-/-) and lacking telomerase activity can only be bred for approximately six generations due to decreased male and female fertility and to an increased embryonic lethality associated with a neural tube closure defect. Although late generation mTR-/- mice show defects in the hematopoietic system, they are viable to adulthood, only showing a decrease in viability in old age. To assess the contribution of genetic background to the effect of telomerase deficiency on viability, we generated mTR-/- mutants on a C57BL6 background, which showed shorter telomeres than the original mixed genetic background C57BL6/129Sv. Interestingly, these mice could be bred for only four generations and the survival of late generation mTR-/- mice decreased dramatically with age as compared with their wild-type counterparts. Fifty percent of the generation 4 mice die at only 5 months of age. This decreased viability with age in the late generation mice is coincident with telomere shortening, sterility, splenic atrophy, reduced proliferative capacity of B and T cells, abnormal hematology and atrophy of the small intestine. These results indicate that telomere shortening in mTR-/- mice leads to progressive loss of organismal viability.

Increased epidermal tumors and increased skin wound healing in transgenic mice overexpressing the catalytic subunit of telomerase, mTERT, in basal keratinocytes.

Telomerase transgenics are an important tool to assess the role of telomerase in cancer, as well as to evaluate the potential use of telomerase for gene therapy of age-associated diseases. Here, we have targeted the expression of the catalytic component of mouse telomerase, mTERT, to basal keratinocytes using the bovine keratin 5 promoter. These telomerase-transgenic mice are viable and show histologically normal stratified epithelia with high levels of telomerase activity and normal telomere length. Interestingly, the epidermis of these mice is highly responsive to the mitogenic effects of phorbol esters, and it is more susceptible than that of wild-type littermates to the development skin tumors upon chemical carcinogenesis. The epidermis of telomerase-transgenic mice also shows an increased wound-healing rate compared with wild-type littermates. These results suggest that, contrary to the general assumption, telomerase actively promotes proliferation in cells that have sufficiently long telomeres and unravel potential risks of gene therapy for age-associated diseases based on telomerase upregulation.

Limited overlapping roles of P15(INK4b) and P18(INK4c) cell cycle inhibitors in proliferation and tumorigenesis.

Entry of quiescent cells into the cell cycle is driven by the cyclin D-dependent kinases Cdk4 and Cdk6. These kinases are negatively regulated by the INK4 cell cycle inhibitors. We report the generation of mice defective in P15(INK4b) and P18(INK4c). Ablation of these genes, either alone or in combination, does not abrogate cell contact inhibition or senescence of mouse embryo fibroblasts in culture. However, loss of P15(INK4b), but not of P18(INK4c), confers proliferative advantage to these cells and makes them more sensitive to transformation by H-ras oncogenes. In vivo, ablation of P15(INK4b) and P18(INK4c) genes results in lymphoproliferative disorders and tumor formation. Mice lacking P18(INK4c) have deregulated epithelial cell growth leading to the formation of cysts, mostly in the cortical region of the kidneys and the mammary epithelium. Loss of both P15(INK4b) and P18(INK4c) does not result in significantly distinct phenotypic manifestations except for the appearance of cysts in additional tissues. These results indicate that P15(INK4b) and P18(IKN4c) are tumor suppressor proteins that act in different cellular lineages and/or pathways with limited compensatory roles.

The ink4a/arf tumor suppressors cooperate with p21cip1/waf in the processes of mouse epidermal differentiation, senescence, and carcinogenesis.

In mammalian cells, cell cycle withdrawal is a prerequisite for terminal differentiation. Accordingly, in most tissues, including epidermis, the expression of the cyclin-dependent kinase inhibitors increases during differentiation. However, the actual role of cyclin-dependent kinase inhibitors is unclear. Different aspects of epidermal growth and differentiation in ink4a(Delta2,3)-null, p21-null, and ink4a(Delta2,3)/p21-doubly deficient mice were studied. Altered differentiation and decreased age-related senescence were found in the epidermis of ink4a(Delta2,3)/p21-null mice and, to a lesser extent, in ink4a(Delta2,3)- and p21-null mice. ink4a(Delta2,3)/p21-null primary keratinocytes underwent cell cycle arrest upon calcium or transforming growth factor-beta treatment, but failed to differentiate. This differentiation deficiency was not observed in p21- or ink4a(Delta2,3)-deficient keratinocytes. Upon infection with a v-Ha-ras-coding retrovirus, wild-type keratinocytes displayed features indicative of premature cell senescence. In p21- or ink4a(Delta2,3)-deficient keratinocytes, only a partial response was observed. ink4a(Delta2,3)/p21-deficient keratinocytes did not display senescent features, but showed increased tumorigenic potential upon injection into nude mice. These results indicate that ink4a/arf and cip1/waf genes cooperate to allow normal keratinocyte differentiation and that the absence of both favors malignant transformation.

[Bladder puncture in premature newborn infants with bacteriuria in the urine collected in bags]. / Punción vesical en neonatos pretérmino con bacteriuria en orina recogida mediante bolsa.

A prospective study was made in a selected group of premature newborns who had presented bacteriuria in bag collected urine. Authors have analysed the real inside of urinary tract infection evidenced by suprapubic bladder aspiration in these children. These study includes the separation in two groups, with or without symptomatology, and therapy used in both cases. A high incidence of false bacteriurias was observed in bag collected urines, early in the asymptomatic group, where females were greater part. They have observed also a small correlation between clinic manifestations and urinocultive results and urinary sediment. The possible clinical, biological and bacteriological parameters were considered in order to see the better to suspect a urinary tract infection. Clinical symptomatology, and suprapubic bladder aspiration were the best to confirm it. Authors don't recommend neither aspiration nor urinocultive as routine techniques in prematures.

Prevalencia de la dermatitis atópica en escolares de Cartagena y su relación con el sexo y la contaminación / PRevalence of atopic dermatitis in schoolchildren from Cartagena (Spain) and relationship with sex and pollution

Objetivo El objetivo del presente trabajo es estudiar la prevalencia de la dermatitis atópica y la influencia del sexo y la contaminación atmosférica de la ciudad de Cartagena. Métodos y diseño Estudio transversal, mediante encuesta de investigación del Estudio ISAAC (International Study of Asthma and Allergies in Childhood), en los escolares de 13 y 14 años de Cartagena (Murcia), analizando el sexo y la ubicación de los colegios a los que acuden estos escolares según dos zonas (contaminada y no), establecidas previamente; y la prevalencia y gravedad del eccema (manifestado por despertar por la noche a causa del prurito), según el sexo y según cada zona analizada. Resultados Los resultados muestran una prevalencia del eccema atópico del 6,3 por ciento, y es grave en una quinta parte de los casos, con mayor prevalencia significativa (odds ratio [OR], 2,19; intervalo de confianza del 95 por ciento [IC 95 por ciento], 1,59-3,02), pero no mayor gravedad, en el sexo femenino, y con mayor prevalencia (OR, 1,83; IC 95 por ciento, 1,01-1,87), pero tampoco mayor gravedad significativas en los escolares que asisten a los colegios en la zona contaminada. Conclusiones La contaminación atmosférica se asocia con una mayor prevalencia de eccema atópico y existe una tendencia de que éste sea más grave. El eccema fue más prevalente entre las niñas (AU)

Asociación entre dermatitis atópica, rinitis alérgica y asma en escolares de 13 y 14 años / Association between atopic dermatitis, allergic rhinitis and asthma in schoolchildren aged 13-14 years old

Antecedentes La dermatitis atópica está en íntima relación con otras enfermedades atópicas, particularmente con asma y rinitis alérgica. A pesar de haber cierto acuerdo en que el padecimiento de eccema atópico predispone al padecimiento de asma y rinitis, es preciso cuantificar el riesgo en las poblaciones, siguiendo un método de estudio estandarizado, que es el objetivo del presente trabajo.Método Siguiendo una metodología de encuesta de investigación del estudio ISAAC, en un estudio transversal, hemos analizado, y cuantificado, en la población de escolares de 13 y 14 años de la ciudad de Cartagena (Murcia), la asociación del padecimiento y gravedad de eccema atópico con el de la rinitis alérgica, el asma y el asma alérgica.Resultados Los resultados han mostrado, cuantificando el riesgo, que los escolares con eccema atópico, respecto a los que no lo padecen, tienen un riesgo triple de tener rinitis alérgica (odds ratio [OR], 3,33; intervalo de confianza del 95 por ciento [IC 95 por ciento], 2,45-4,54), cuádruple de tener asma (OR, 3,85; IC 95 por ciento, 2,74-5,42) y quíntuple de tener asma alérgica (OR, 4,91; IC 95 por ciento, 3,17-7,59), estando en relación directa las gravedades respectivas (AU)