Reprogramming in vivo produces teratomas and iPS cells with totipotency features.

Reprogramming of adult cells to generate induced pluripotent stem cells (iPS cells) has opened new therapeutic opportunities; however, little is known about the possibility of in vivo reprogramming within tissues. Here we show that transitory induction of the four factors Oct4, Sox2, Klf4 and c-Myc in mice results in teratomas emerging from multiple organs, implying that full reprogramming can occur in vivo. Analyses of the stomach, intestine, pancreas and kidney reveal groups of dedifferentiated cells that express the pluripotency marker NANOG, indicative of in situ reprogramming. By bone marrow transplantation, we demonstrate that haematopoietic cells can also be reprogrammed in vivo. Notably, reprogrammable mice present circulating iPS cells in the blood and, at the transcriptome level, these in vivo generated iPS cells are closer to embryonic stem cells (ES cells) than standard in vitro generated iPS cells. Moreover, in vivo iPS cells efficiently contribute to the trophectoderm lineage, suggesting that they achieve a more plastic or primitive state than ES cells. Finally, intraperitoneal injection of in vivo iPS cells generates embryo-like structures that express embryonic and extraembryonic markers. We conclude that reprogramming in vivo is feasible and confers totipotency features absent in standard iPS or ES cells. These discoveries could be relevant for future applications of reprogramming in regenerative medicine.

Fatty acids homeostasis during fasting predicts protection from chemotherapy toxicity.

Fasting exerts beneficial effects in mice and humans, including protection from chemotherapy toxicity. To explore the involved mechanisms, we collect blood from humans and mice before and after 36 or 24 hours of fasting, respectively, and measure lipid composition of erythrocyte membranes, circulating micro RNAs (miRNAs), and RNA expression at peripheral blood mononuclear cells (PBMCs). Fasting coordinately affects the proportion of polyunsaturated versus saturated and monounsaturated fatty acids at the erythrocyte membrane; and reduces the expression of insulin signaling-related genes in PBMCs. When fasted for 24 hours before and 24 hours after administration of oxaliplatin or doxorubicin, mice show a strong protection from toxicity in several tissues. Erythrocyte membrane lipids and PBMC gene expression define two separate groups of individuals that accurately predict a differential protection from chemotherapy toxicity, with important clinical implications. Our results reveal a mechanism of fasting associated with lipid homeostasis, and provide biomarkers of fasting to predict fasting-mediated protection from chemotherapy toxicity.

Activation of p21 limits acute lung injury and induces early senescence after acid aspiration and mechanical ventilation.

The p53/p21 pathway is activated in response to cell stress. However, its role in acute lung injury has not been elucidated. Acute lung injury is associated with disruption of the alveolo-capillary barrier leading to acute respiratory distress syndrome (ARDS). Mechanical ventilation may be necessary to support gas exchange in patients with ARDS, however, high positive airway pressures can cause regional overdistension of alveolar units and aggravate lung injury. Here, we report that acute lung injury and alveolar overstretching activate the p53/p21 pathway to maintain homeostasis and avoid massive cell apoptosis. A systematic pooling of transcriptomic data from animal models of lung injury demonstrates the enrichment of specific p53- and p21-dependent gene signatures and a validated senescence profile. In a clinically relevant, murine model of acid aspiration and mechanical ventilation, we observed changes in the nuclear envelope and the underlying chromatin, DNA damage and activation of the Tp53/p21 pathway. Absence of Cdkn1a decreased the senescent response, but worsened lung injury due to increased cell apoptosis. Conversely, treatment with lopinavir and/or ritonavir led to Cdkn1a overexpression and ameliorated cell apoptosis and lung injury. The activation of these mechanisms was associated with early markers of senescence, including expression of senescence-related genes and increases in senescence-associated heterochromatin foci in alveolar cells. Autopsy samples from lungs of patients with ARDS revealed increased senescence-associated heterochromatin foci. Collectively, these results suggest that acute lung injury activates p53/p21 as an antiapoptotic mechanism to ameliorate damage, but with the side effect of induction of senescence.

Senescence promotes in vivo reprogramming through p16INK4a and IL-6.

Cellular senescence is a damage response aimed to orchestrate tissue repair. We have recently reported that cellular senescence, through the paracrine release of interleukin-6 (IL6) and other soluble factors, strongly favors cellular reprogramming by Oct4, Sox2, Klf4, and c-Myc (OSKM) in nonsenescent cells. Indeed, activation of OSKM in mouse tissues triggers senescence in some cells and reprogramming in other cells, both processes occurring concomitantly and in close proximity. In this system, Ink4a/Arf-null tissues cannot undergo senescence, fail to produce IL6, and cannot reprogram efficiently; whereas p53-null tissues undergo extensive damage and senescence, produce high levels of IL6, and reprogram efficiently. Here, we have further explored the genetic determinants of in vivo reprogramming. We report that Ink4a, but not Arf, is necessary for OSKM-induced senescence and, thereby, for the paracrine stimulation of reprogramming. However, in the absence of p53, IL6 production and reprogramming become independent of Ink4a, as revealed by the analysis of Ink4a/Arf/p53 deficient mice. In the case of the cell cycle inhibitor p21, its protein levels are highly elevated upon OSKM activation in a p53-independent manner, and we show that p21-null tissues present increased levels of senescence, IL6, and reprogramming. We also report that Il6-mutant tissues are impaired in undergoing reprogramming, thus reinforcing the critical role of IL6 in reprogramming. Finally, young female mice present lower efficiency of in vivo reprogramming compared to male mice, and this gender difference disappears with aging, both observations being consistent with the known anti-inflammatory effect of estrogens. The current findings regarding the interplay between senescence and reprogramming may conceivably apply to other contexts of tissue damage.

Inactivation of imprinted genes induced by cellular stress and tumorigenesis.

Cellular proliferation under stressful conditions may result in permanent genetic and epigenetic changes. Using primary mouse embryonic fibroblasts, we have completed a screening test to identify gene expression changes triggered when cells proliferate under stress. In this manner, we have discovered a novel phenomenon that consists of the rapid and coordinated silencing of genes subject to imprinting, including Cdkn1c, Igf2, H19, Ndn1, Grb10, and Meg3. This generalized silencing of imprinted genes is independent of the stress-responsive tumor suppressors p53, p19(Arf), and p16(Ink4a), and it is also independent of the oxidative culture conditions and the stress response known as "mouse embryonic fibroblast senescence". In the case of Cdkn1c and H19, their silencing is associated with unscheduled de novo methylation of the normally expressed allele at their corresponding CpG island promoters, thus resulting in biallelic methylation. Finally, we provide evidence for frequent de novo methylation of Cdkn1c in a variety of murine cancer types. Altogether, our data support the concept that silencing of imprinted genes, including methylation of Cdkn1c, constitutes an epigenetic signature of cellular stress and tumorigenesis.

p21Cip1 plays a critical role in the physiological adaptation to fasting through activation of PPARα.

Fasting is a physiological stress that elicits well-known metabolic adaptations, however, little is known about the role of stress-responsive tumor suppressors in fasting. Here, we have examined the expression of several tumor suppressors upon fasting in mice. Interestingly, p21 mRNA is uniquely induced in all the tissues tested, particularly in liver and muscle (>10 fold), and this upregulation is independent of p53. Remarkably, in contrast to wild-type mice, p21-null mice become severely morbid after prolonged fasting. The defective adaptation to fasting of p21-null mice is associated to elevated energy expenditure, accelerated depletion of fat stores, and premature activation of protein catabolism in the muscle. Analysis of the liver transcriptome and cell-based assays revealed that the absence of p21 partially impairs the transcriptional program of PPARα, a key regulator of fasting metabolism. Finally, treatment of p21-null mice with a PPARα agonist substantially protects them from their accelerated loss of fat upon fasting. We conclude that p21 plays a relevant role in fasting adaptation through the positive regulation of PPARα.

Tissue damage and senescence provide critical signals for cellular reprogramming in vivo.

Reprogramming of differentiated cells into pluripotent cells can occur in vivo, but the mechanisms involved remain to be elucidated. Senescence is a cellular response to damage, characterized by abundant production of cytokines and other secreted factors that, together with the recruitment of inflammatory cells, result in tissue remodeling. Here, we show that in vivo expression of the reprogramming factors OCT4, SOX2, KLF4, and cMYC (OSKM) in mice leads to senescence and reprogramming, both coexisting in close proximity. Genetic and pharmacological analyses indicate that OSKM-induced senescence requires the Ink4a/Arf locus and, through the production of the cytokine interleukin-6, creates a permissive tissue environment for in vivo reprogramming. Biological conditions linked to senescence, such as tissue injury or aging, favor in vivo reprogramming by OSKM. These observations may be relevant for tissue repair.

Activation of cyclin D1-kinase in murine fibroblasts lacking both p21(Cip1) and p27(Kip1).

Deregulation of D-type cyclin-dependent kinases (CDK4 and 6) is widely observed in various human cancers, illustrating their importance in cell cycle control. Like other cyclin-dependent kinases (CDKs), assembly with cyclins is the most critical step for activation of CDK4/6. As previously reported elsewhere, we observed that the level of cyclinD1-CDK4 complex and its associated kinase activity were significantly low in asynchronously proliferating mouse embryo fibroblasts lacking both p21(Cip1) and p27(Kip1) (p21/p27-null MEFs). These evidences imply that p21(Cip1) and p27(Kip1) CDK inhibitors are 'essential activators' of cyclin D-kinases. We, however, discovered here that both the assembly and activation of cyclin D1-CDK4 complex occur when quiescent p21/p27-null MEFs were stimulated to re-enter the cell cycle. This mitogen-induced cyclin D1-kinase activity was blocked by overexpression of p16(INK4a) and resulted in the inhibition of S phase entry in p21/p27-null MEFs. Furthermore, ectopic expression of p34(SEI-1), a mitogen-induced CDK4 binding protein, increased the levels of active cyclinD1-CDK4 complex in asynchronously proliferating p21/p27-null MEFs. Together, our results suggest that there are several independent ways to stimulate the assembly of cyclin D1-CDK4 kinases. Although p21(Cip1) and p27(Kip1) play a role in this process, our results demonstrate that additional mechanisms must occur in G0 to S phase transition.

Lineage-restricted function of the pluripotency factor NANOG in stratified epithelia.

NANOG is a pluripotency transcription factor in embryonic stem cells; however, its role in adult tissues remains largely unexplored. Here we show that mouse NANOG is selectively expressed in stratified epithelia, most notably in the oesophagus where the Nanog promoter is hypomethylated. Interestingly, inducible ubiquitous overexpression of NANOG in mice causes hyperplasia selectively in the oesophagus, in association with increased cell proliferation. NANOG transcriptionally activates the mitotic programme, including Aurora A kinase (Aurka), in stratified epithelia, and endogenous NANOG directly binds to the Aurka promoter in primary keratinocytes. Interestingly, overexpression of Nanog or Aurka in mice increased proliferation and aneuploidy in the oesophageal basal epithelium. Finally, inactivation of NANOG in cell lines from oesophageal or head and neck squamous cell carcinomas (ESCCs or HNSCCs, respectively) results in lower levels of AURKA and decreased proliferation, and NANOG and AURKA expression are positively correlated in HNSCCs. Together, these results indicate that NANOG has a lineage-restricted mitogenic function in stratified epithelia.

Genome-wide CTCF distribution in vertebrates defines equivalent sites that aid the identification of disease-associated genes.

Many genomic alterations associated with human diseases localize in noncoding regulatory elements located far from the promoters they regulate, making it challenging to link noncoding mutations or risk-associated variants with target genes. The range of action of a given set of enhancers is thought to be defined by insulator elements bound by the 11 zinc-finger nuclear factor CCCTC-binding protein (CTCF). Here we analyzed the genomic distribution of CTCF in various human, mouse and chicken cell types, demonstrating the existence of evolutionarily conserved CTCF-bound sites beyond mammals. These sites preferentially flank transcription factor-encoding genes, often associated with human diseases, and function as enhancer blockers in vivo, suggesting that they act as evolutionarily invariant gene boundaries. We then applied this concept to predict and functionally demonstrate that the polymorphic variants associated with multiple sclerosis located within the EVI5 gene impinge on the adjacent gene GFI1.

Normal proliferation and tumorigenesis but impaired pancreatic function in mice lacking the cell cycle regulator sei1.

Sei1 is a positive regulator of proliferation that promotes the assembly of Cdk4-cyclin D complexes and enhances the transcriptional activity of E2f1. The potential oncogenic role of Sei1 is further suggested by its overexpression in various types of human cancers. To study the role of Sei1, we have generated a mouse line deficient for this gene. Sei1-null fibroblasts did not show abnormalities regarding proliferation or susceptibility to neoplastic transformation, nor did we observe defects on Cdk4 complexes or E2f activity. Sei1-null mice were viable, did not present overt pathologies, had a normal lifespan, and had a normal susceptibility to spontaneous and chemically-induced cancer. Pancreatic insulin-producing cells are known to be particularly sensitive to Cdk4-cyclin D and E2f activities, and we have observed that Sei1 is highly expressed in pancreatic islets compared to other tissues. Interestingly, Sei1-null mice present lower number of islets, decreased beta-cell area, impaired insulin secretion, and glucose intolerance. These defects were associated to nuclear accumulation of the cell-cycle inhibitors p21(Cip1) and p27(Kip1) in islet cells. We conclude that Sei1 plays an important role in pancreatic beta-cells, which supports a functional link between Sei1 and the core cell cycle regulators specifically in the context of the pancreas.

[Determination of insecticide-resistance and resistance mechanisms of Blattella germanica (Dictyoptera: Blattellidae)]. / Determinación de la resistencia a insecticidas y mecanismos de resistencia en cepas de Blattella germanica (Dictyoptera: Blattellidae).

In this paper, the level of resistance to four insecticides of 3 Blatella germanica strains collected from various places in the City of Havana province was evaluated. These strains were resistant to two pyrethroids (cypermethrin and lambda-cyalothrine) and to organophosphorate malathion but susceptible to carbamate propoxur. The values of alpha and beta esterases, acetylcholinesterase and gluthatione-S-transferase were estimated in three strains involved in the study. The results of the study showed high esterase activity in all the strains, mainly beta esterases and two of the three strains presented with high gluthation-S-transferase enzyme. No changes in acetylcholinesterase were demonstrated in relation to the reference strain. The association of levels of resistance to insecticides, the possible resistance mechanisms in each strain and the results of the enzymatic activity were also analyzed.

[Comprehensive surveillance system for dengue prevention]. / Sistema integrado de vigilancia para la prevención de dengue.

A comprehensive ecosystem approach-based surveillance system was designed and implemented for dengue prevention in Cotorro municipality at the City of Havana. Cuba, geographically situated near those countries with high dengue incidence, and having a preventive approach as a premise of its public healthcare system, has to adopt measures to prevent new dengue epidemics. The environmental, entomological and clinical/epidemiological elements of the surveillance system were integrated and combined with a social participation strategy. A number of workshops were held for the people involved in search and analysis of the collected information. An automated database with indicators and thematic map outputs made risk stratification for dengue and its vector possible. Additionally, 17 groups of neighbors were organized. The environmental surveillance was the first element to be taken into account to avoid Aedes aegypti spread. The comprehensive surveillance system for dengue developed in the project was an important tool for the decision-taking process at local level.

Specific contribution of p19(ARF) to nitric oxide-dependent apoptosis.

NO is an important bioactive molecule involved in a variety of physio- and pathological processes, including apoptosis induction. The proapoptotic activity of NO involves the rise in the tumor suppressor p53 and the accumulation and targeting of proapoptotic members of the Bcl-2 family, in particular Bax and the release of cytochrome c from the mitochondria. However, the exact mechanism by which NO induces p53 activation has not been fully elucidated. In this study, we describe that NO induces p19(ARF) through a transcriptional mechanism. This up-regulation of p19(ARF) activates p53, leading to apoptosis. The importance of p19(ARF) on NO-dependent apoptosis was revealed by the finding that various cell types from alternate reading frame-knockout mice exhibit a diminished response to NO-mediated apoptosis when compared with normal mice. Moreover, the biological relevance of alternative reading frame to p53 apoptosis was confirmed in in vivo models of apoptosis. Together, these results demonstrate that NO-dependent apoptosis requires, in part, the activation of p19(ARF).

Increased gene dosage of Ink4a/Arf results in cancer resistance and normal aging.

Mammalian genes frequently present allelic variants that differ in their expression levels and that, in the case of tumor suppressor genes, can be of relevance for cancer susceptibility and aging. We report here the characterization of a novel mouse model with increased activity for the Ink4a and Arf tumor suppressors. We have generated a "super Ink4a/Arf" mouse strain carrying a transgenic copy of the entire Ink4a/Arf locus. Cells derived from super Ink4a/Arf mice have increased resistance to in vitro immortalization and oncogenic transformation. Importantly, super Ink4a/Arf mice manifest higher resistance to cancer compared to normal, nontransgenic, mice. Finally, super Ink4a/Arf mice have normal aging and lifespan. Together, these results indicate that modest increases in the activity of the Ink4a/Arf tumor suppressor result in a beneficial cancer-resistant phenotype without affecting normal viability or aging.

The p21(Cip1) protein, a cyclin inhibitor, regulates the levels and the intracellular localization of CDC25A in mice regenerating livers.

Liver cells from p21(Cip1-/-) mice subjected to partial hepatectomy (PH) progress into DNA synthesis faster than those from wild-type mice. These cells also show a premature induction of cyclin E/cyclin-dependent kinase (CDK) 2 activity. We studied the mechanisms whereby cells lacking p21(Cip1) showed a premature induction of this activity. Whereas the levels of CDK2, cyclin E, and p27(Kip1) were similar in both wild-type and p21(Cip1-/-) mice, those of the activator CDC25A were much higher in p21(Cip1-/-) quiescent and regenerating livers than in wild-type animals. Moreover, p21(Cip1-/-) cells also showed a premature translocation of CDC25A from cytoplasm into the nucleus. The ectopic expression of p21(Cip1) into mice embryo fibroblasts from p21(Cip1-/-) mice decreased the levels of CDC25A and delayed its nuclear translocation. The levels of CDC25A messenger RNA in p21(Cip1-/-) cells were higher than in wild-type cells, suggesting that this increase might be responsible, at least in part, for the high levels of CDC25A protein in these cells. Thus, the results reported here indicate that p21(Cip1) regulates the levels and the intracellular localization of CDC25A. We also found a good correlation between CDC25A nuclear translocation and cyclin E/CDK2 activation. In conclusion, premature translocation of CDC25A to the nucleus might be involved in the advanced induction of cyclin E/CDK2 activity and DNA replication in cells from animals lacking p21(Cip1).

Determinación de la resistencia a insecticidas y mecanismos de resistencia en cepas de Blattella germanica (Dictyoptera: Blattellidae) / Determination of insecticide-resistance and resistance mechanisms of Blattella germanica (Dictyoptera: Blattellidae)

Se evaluaron los niveles de resistencia en 3 cepas de Blattella germanica colectadas en diferentes lugares de Ciudad de La Habana, frente a 4 insecticidas. Las cepas fueron resistentes a los 2 piretroides evaluados (cipermetrina y lambdacialotrina) y al organofosforado malatión, mostrándose susceptibles ante el carbamato propoxur. Se determinaron los valores α y ß Esterasas, acetilcolinesterasa y glutation S-transferasa, a ejemplares de las 3 cepas involucradas en el estudio. Los resultados del trabajo mostraron una elevada actividad de esterasas en todas las cepas, sobre todo de las ß Esterasas, en 2 de las 3 cepas estudiadas la enzima glutation S-transferasa fue elevada y no se demostró que existan modificaciones en la acetilcolinesterasa en relación con la cepa de referencia. La relación entre los niveles de resistencia a insecticidas, los posibles mecanismos de resistencia presentes en cada cepa y los resultados de la actividad enzimática, fueron analizados.

In this paper, the level of resistance to four insecticides of 3 Blatella germanica strains collected from various places in the City of Havana province was evaluated. These strains were resistant to two pyrethroids (cypermethrin and lambdacyalothrine) and to organophosphorate malathion but susceptible to carbamate propoxur. The values of α and ß esterases, acetylcholinesterase and gluthatione-S-transferase were estimated in three strains involved in the study. The results of the study showed high esterase activity in all the strains, mainly ß esterases and two of the three strains presented with high gluthation-S-transferase enzyme. No changes in acetylcholinesterase were demonstrated in relation to the reference strain. The association of levels of resistance to insecticides, the possible resistance mechanisms in each strain and the results of the enzymatic activity were also analyzed.

Determinación de la resistencia a insecticidas y mecanismos de resistencia en cepas de Blattella germanica (Dictyoptera: Blattellidae) / Determination of insecticide-resistance and resistance mechanisms of Blattella germanica (Dictyoptera: Blattellidae)

Se evaluaron los niveles de resistencia en 3 cepas de Blattella germanica colectadas en diferentes lugares de Ciudad de La Habana, frente a 4 insecticidas. Las cepas fueron resistentes a los 2 piretroides evaluados (cipermetrina y lambdacialotrina) y al organofosforado malatión, mostrándose susceptibles ante el carbamato propoxur. Se determinaron los valores α y ß Esterasas, acetilcolinesterasa y glutation S-transferasa, a ejemplares de las 3 cepas involucradas en el estudio. Los resultados del trabajo mostraron una elevada actividad de esterasas en todas las cepas, sobre todo de las ß Esterasas, en 2 de las 3 cepas estudiadas la enzima glutation S-transferasa fue elevada y no se demostró que existan modificaciones en la acetilcolinesterasa en relación con la cepa de referencia. La relación entre los niveles de resistencia a insecticidas, los posibles mecanismos de resistencia presentes en cada cepa y los resultados de la actividad enzimática, fueron analizados(AU)

In this paper, the level of resistance to four insecticides of 3 Blatella germanica strains collected from various places in the City of Havana province was evaluated. These strains were resistant to two pyrethroids (cypermethrin and lambdacyalothrine) and to organophosphorate malathion but susceptible to carbamate propoxur. The values of α and ß esterases, acetylcholinesterase and gluthatione-S-transferase were estimated in three strains involved in the study. The results of the study showed high esterase activity in all the strains, mainly ß esterases and two of the three strains presented with high gluthation-S-transferase enzyme. No changes in acetylcholinesterase were demonstrated in relation to the reference strain. The association of levels of resistance to insecticides, the possible resistance mechanisms in each strain and the results of the enzymatic activity were also analyzed(AU)