Casein kinase 2 phosphorylates and induces the SALL2 tumor suppressor degradation in colon cancer cells.

Spalt-like proteins are Zinc finger transcription factors from Caenorhabditis elegans to vertebrates, with critical roles in development. In vertebrates, four paralogues have been identified (SALL1-4), and SALL2 is the family's most dissimilar member. SALL2 is required during brain and eye development. It is downregulated in cancer and acts as a tumor suppressor, promoting cell cycle arrest and cell death. Despite its critical functions, information about SALL2 regulation is scarce. Public data indicate that SALL2 is ubiquitinated and phosphorylated in several residues along the protein, but the mechanisms, biological consequences, and enzymes responsible for these modifications remain unknown. Bioinformatic analyses identified several putative phosphorylation sites for Casein Kinase II (CK2) located within a highly conserved C-terminal PEST degradation motif of SALL2. CK2 is a serine/threonine kinase that promotes cell proliferation and survival and is often hyperactivated in cancer. We demonstrated that CK2 phosphorylates SALL2 residues S763, T778, S802, and S806 and promotes SALL2 degradation by the proteasome. Accordingly, pharmacological inhibition of CK2 with Silmitasertib (CX-4945) restored endogenous SALL2 protein levels in SALL2-deficient breast MDA-MB-231, lung H1299, and colon SW480 cancer cells. Silmitasertib induced a methuosis-like phenotype and cell death in SW480 cells. However, the phenotype was significantly attenuated in CRISPr/Cas9-mediated SALL2 knockout SW480 cells. Similarly, Sall2-deficient tumor organoids were more resistant to Silmitasertib-induced cell death, confirming that SALL2 sensitizes cancer cells to CK2 inhibition. We identified a novel CK2-dependent mechanism for SALL2 regulation and provided new insights into the interplay between these two proteins and their role in cell survival and proliferation.

Streamlined computational pipeline for genetic background characterization of genetically engineered mice based on next generation sequencing data.

BACKGROUND: Genetically engineered mice (GEM) are essential tools for understanding gene function and disease modeling. Historically, gene targeting was first done in embryonic stem cells (ESCs) derived from the 129 family of inbred strains, leading to a mixed background or congenic mice when crossed with C57BL/6 mice. Depending on the number of backcrosses and breeding strategies, genomic segments from 129-derived ESCs can be introgressed into the C57BL/6 genome, establishing a unique genetic makeup that needs characterization in order to obtain valid conclusions from experiments using GEM lines. Currently, SNP genotyping is used to detect the extent of 129-derived ESC genome introgression into C57BL/6 recipients; however, it fails to detect novel/rare variants. RESULTS: Here, we present a computational pipeline implemented in the Galaxy platform and in BASH/R script to determine genetic introgression of GEM using next generation sequencing data (NGS), such as whole genome sequencing (WGS), whole exome sequencing (WES) and RNA-Seq. The pipeline includes strategies to uncover variants linked to a targeted locus, genome-wide variant visualization, and the identification of potential modifier genes. Although these methods apply to congenic mice, they can also be used to describe variants fixed by genetic drift. As a proof of principle, we analyzed publicly available RNA-Seq data from five congenic knockout (KO) lines and our own RNA-Seq data from the Sall2 KO line. Additionally, we performed target validation using several genetics approaches. CONCLUSIONS: We revealed the impact of the 129-derived ESC genome introgression on gene expression, predicted potential modifier genes, and identified potential phenotypic interference in KO lines. Our results demonstrate that our new approach is an effective method to determine genetic introgression of GEM.

Sall2 is required for proapoptotic Noxa expression and genotoxic stress-induced apoptosis by doxorubicin.

The Sall2 transcription factor is deregulated in several cancers; however, little is known about its cellular functions, including its target genes. Recently, we demonstrated that p53 directly regulates Sall2 expression under genotoxic stress. Here, we investigated the role of Sall2 in the context of cellular response to genotoxic stress. In addition, we further examined the Sall2-p53 relationship during genotoxic stress in primary mouse embryo fibroblasts (MEFs), which are derived from Sall2 knockout mice separately, or in combination with the p53ERTAM knock-in mice. We found that the levels of Sall2 mRNA and protein are dynamically modulated in response to doxorubicin. At early times of stress, Sall2 is downregulated, but increases under extension of the stress in a p53-independent manner. Based on caspase-3/7 activities, expression of cleaved poly (ADP-ribose) polymerase, expression of cleaved caspase-3 and induction of proapoptotic proteins, Sall2 expression was correlated with cellular apoptosis. Consequently, Sall2-/- MEFs have decreased apoptosis, which relates with increased cell viability in response to doxorubicin. Importantly, Sall2 was required for apoptosis even in the presence of fully activated p53. Searching for putative Sall2 targets that could mediate its role in apoptosis, we identified proapoptotic NOXA/PMAIP1 (phorbol-12-myristate-13-acetate-induced protein 1). We demonstrated that Sall2 positively regulates Noxa promoter activity. Conserved putative Sall2-binding sites at the NOXA promoter were validated in vitro by electrophoretic mobility shift assay and in vivo by ChIP experiments, identifying NOXA as a novel Sall2 target. In agreement, induction of Noxa protein and mRNA in response to doxorubicin was significantly decreased in Sall2-/- MEFs. In addition, studies in leukemia Jurkat T cells support the existence of the Sall2/Noxa axis, and the significance of this axis on the apoptotic response to doxorubicin in cancer cells. Our study highlights the relevance of Sall2 in the apoptotic response to extended genotoxic stress, which is important for understanding its role in normal physiology and disease.

Violencia escolar y su relación con síntomas ansiosos / Bullying and its association with anxiety symptoms

INTRODUCCIÓN: Violencia escolar es la persecución física o psicológica de un alumno o alumna contra otro, convirtiéndolo en víctima de repetidos ataques. Es un importante marcador de riesgo de conductas antisociales futuras, y se relaciona con mayor prevalencia de síntomas ansiosos. OBJETIVO: Establecer si existe asociación entre la presencia de ansiedad patológica y la práctica de violencia escolar. MATERIAL Y MÉTODOS: Estudio descriptivo observacional y transversal. Se estudió a 166 alumnos de cuarto a octavo básico, de un colegio particular subvencionado de la comuna de Lo Barnechea, en Santiago de Chile. De ellos, 77 fueron hombres y 89 mujeres, entre 9 y 16 años. Se aplicó la escala de Autoreporte de Ansiedad para Niños y Adolescentes (AANA) y el cuestionario de Maltrato entre Iguales por Abuso de Poder (MIAP). Además, se recopilaron datos tales como sexo, edad, número de personas que viven en el hogar y si vive o no con sus padres. RESULTADOS: De los estudiantes evaluados, el 46,99 por ciento clasificó como testigo de violencia escolar, 19,28 por ciento víctima, 10,84 por ciento agresor, 10,84 por ciento víctima-agresor, y un 12,05 por ciento no clasificó en ninguna categoría. Se obtuvo un 36,1 por ciento de prevalencia de ansiedad patológica en el total de individuos; un 41,67 por ciento en agresores y 30,61 por ciento en no agresores, con chi-cuadrado P > 0,05.DISCUSIÓN: La prevalencia de ansiedad patológica en agresores de violencia escolar es mayor que en quienes no la practican; sin embargo, esta asociación no llega a valores estadísticamente significativos.

INTRODUCTION: Bullying is the physical or psychological persecution of one student against another, making him a victim of recurrent attacks. It is one of the most important risk markers for future antisocial behavior, which has also been associated with higher prevalence of anxiety symptoms. OBJECTIVE: Establish if there is an association between pathological anxiety and bullying. METHODS: This is a descriptive, observational and transversal study. 166 students from fourth to eight grades were studied from a semi private school from Lo Barnechea, Santiago, Chile. From this population, 77 were male and89 female, between 9 and 16 years old. The Anxiety Scale for Children and Adolescents (AAA) and the Survey of High School Bullying Abuse of Power questionnaires were applied. Also, data such as sex, age, number of people who are living in their homes and whether they live with their parents or not was collected. RESULTS: From the students evaluated, 46.99 percent classified as bullying witnesses, 19.28 percent as victims, 10.84 percent as aggressors, 10.84 percent aggressor-victim and 12.05 percent didn't classify in any category. A 36.1 percent prevalence of pathologic anxiety was obtained from the totality of individuals. In the aggressor category 41.67 percent had pathological anxiety and 30.61 percent in non-aggressors, with a chi-square P > 0.05. DISCUSSION: Prevalence of pathological anxiety is higher in the aggressor than in the non-aggressor group, however this association is statistically non-significant

Rheb activates AMPK and reduces p27Kip1 levels in Tsc2-null cells via mTORC1-independent mechanisms: implications for cell proliferation and tumorigenesis.

Tuberous sclerosis complex (TSC) is an autosomally inherited disorder that causes tumors to form in many organs. It is frequently caused by inactivating mutations in the TSC2 tumor-suppressor gene. TSC2 negatively regulates the activity of the GTPase Rheb and thereby inhibits mammalian target of rapamycin complex 1 (mTORC1) signaling. Activation of mTORC1 as a result of lack of TSC2 function is observed in TSC and sporadic lymphangioleiomyomatosis (LAM). TSC2 deficiency has recently been associated with elevated AMP-activated protein kinase (AMPK) activity, which in turn correlated with cytoplasmic localization of p27Kip1 (p27), a negative regulator of cyclin-dependent kinase 2 (Cdk2). How AMPK in the absence of TSC2 is stimulated is not fully understood. In this study, we demonstrate that Rheb activates AMPK and reduces p27 levels in Tsc2-null cells. Importantly, both effects occur largely independent of mTORC1. Furthermore, increased p27 levels following Rheb depletion correlated with reduced Cdk2 activity and cell proliferation in vitro, and with inhibition of tumor formation by Tsc2-null cells in vivo. Taken together, our data suggest that Rheb controls proliferation of TSC2-deficient cells by a mechanism that involves regulation of AMPK and p27, and that Rheb is a potential target for TSC/LAM therapy.

Two subcellular localizations of eIF3 p170 and its interaction with membrane-bound microfilaments: implications for alternative functions of p170.

We previously identified a 170-kDa protein (p170) highly expressed in lung cancers as the major subunit of the eukaryotic translation initiation factor 3 (eIF3). p170 was recently cloned and little is known concerning its characteristics and subcellular localization. In this paper, we report our surprising findings that about 20% of p170 is associated with membranes while the remaining portion is located in the cytoplasm presumably in the eIF3 complex. We also find that p170 interacts with both endoplasmic reticulum and plasma membranes. The binding of p170 to membranes is through actin filaments, consistent with the fact that p170 contains a spectrin repeat motif that may be involved in actin binding. Furthermore, the cytoplasmic p170 is phosphorylated at serine and threonine residues and the phosphorylation is stimulated by serum. However, the membrane-actin-bound p170 is not phosphorylated. The results obtained in this study suggest that p170 may have other functions in addition to participating in translation initiation. Phosphorylation may play an important regulatory role in the function of p170 in translation initiation and other alternative functions.

Identification of a 170-kDa protein over-expressed in lung cancers.

Lung cancer is the leading cause for cancer death in both male and female populations. Although many molecular markers for lung cancer have been developed and useful for early detection of lung cancer, their function remains unknown. In this paper, we report our findings that a 170-kDa protein (p170) is over-expressed in all types of human lung cancers compared with normal tissues and it is identified as a subunit of translation initiation factor eIF3 by cDNA cloning. Translation initiation factors are a family of proteins that promote the initiation step of protein synthesis and are regulators of cell growth at the translational level. Further studies showed that p170 mRNA is ubiquitously expressed with higher levels in adult proliferating tissues (e.g. bone marrow) and tissues during development (e.g. fetal tissues). This study suggests that p170 and eIF3 may be important factors for cell growth, development, and tumorigenesis.

Vascular endothelial cell growth factor activates CRE-binding protein by signaling through the KDR receptor tyrosine kinase.

Vascular endothelial cell growth factor (VEGF) plays a crucial role in the development of the cardiovascular system and in promoting angiogenesis associated with physiological and pathological processes. Although a great deal is known of the cytoplasmic signaling pathways activated by VEGF, much less is known of the mechanisms through which VEGF communicates with the nucleus and alters the activity of transcription factors. Binding of VEGF to the KDR/Flk1 receptor tyrosine kinase induces phosphorylation of the CRE-binding protein (CREB) transcription factor on serine 133 and increases CREB DNA binding and transactivation. p38 MAPK/MSK-1 and protein kinase C/p90RSK pathways mediate CREB phosphorylation. Confocal microscopy shows that VEGF-induced phosphorylation of nuclear CREB is blocked by pharmacological inhibition of protein kinase C and p38 mitogen-activated protein kinase signaling. Thus, KDR/Flk1 uses multiple pathways to transmit signals into the nucleus where CREB becomes activated. These results suggest that CREB may play a role in alterations of gene expression important to angiogenesis.

Increased expression of c-rel, from the NF-kappaB/Rel family, in T cells from patients with systemic lupus erythematosus.

OBJECTIVE: To explore the role of the NF-kappaB/Rel transcription factor family in autoimmunity, we investigated whether peripheral blood mononuclear cells (PBMC) and T cells from the blood of patients with systemic lupus erythematosus (SLE) exhibit abnormal expression of c-rel, both when recently isolated and/or during in vitro activation. METHODS: Total RNA and protein extracts were prepared from PBMC and T cells isolated by immunoadsorption with magnetic beads. The relative concentrations of c-rel mRNA and of c-Rel protein were determined by semiquantitative assays of competitive reverse transcriptase-polymerase chain reaction and chemiluminescent immunoblots, respectively. Activity of NF-kappaB/Rel was studied by electrophoretic mobility shift assay of nuclear extracts. RESULTS: Significantly increased levels of c-rel mRNA were found (1) in PBMC from SLE patients (n = 48; p<0.0000001), even during inactive disease (n = 11; p<0.001), compared to controls (n = 54), and (2) in T cells isolated from a subgroup of these patients (n = 11; p<0.00002) and controls (n = 12). c-Rel protein was found increased in the cytosol but not in the nucleus of PBMC of patients with SLE (n = 12; p<0.02) compared to controls (n = 12). No evidence of NF-kappaB/Rel nuclear activity was detected. In vitro stimulation of T cells by incubating PBMC with concanavalin A showed that less c-Rel entered the nucleus in lupus cells than healthy cells, correlating with lower interleukin 2 production. However, the same stimulating conditions provoked an increase in c-rel mRNA to higher levels in lupus cells from 2 patients compared with 2 controls. Increased levels of both IkappaB alpha and IkappaB beta could account for c-Rel cytosolic retention. CONCLUSION: Our data suggest that T cells from patients with SLE possess altered regulatory mechanisms of c-rel expression and nuclear import that might potentially determine conditions for developing autoimmunity. Other cells present in the PBMC could also be affected.

Clinical evaluation of the effect of calcium-channel blockers on verbal learning.

The effect of verapamil and nimodipine on verbal learning was evaluated in a double-blind clinical trial. Thirty-seven healthy volunteers were distributed in three groups to receive a treatment with nimodipine, verapamil or placebo. Neither verapamil nor nimodipine modifies verbal learning as measured by the selective remembering test of Buschke and Fuld.

Dissection of drug-binding-induced conformational changes in P-glycoprotein.

P-glycoprotein (Pgp) is a membrane-transport ATPase. It uses energy from ATP hydrolysis to transport pleiotropic cytotoxic drugs from inside to outside of cells. Thus, elevated expression of Pgp in cancer cells causes multidrug resistance. It is now known that the conformational state of Pgp changes during its catalytic cycle. However, how ATP hydrolysis relates to drug binding by Pgp is yet to be determined. In this study, we used limited trypsin digestion of Pgp in isolated inside-out membrane vesicles to investigate the effects of drugs on Pgp conformation and to determine the drug-bound conformational states of Pgp in the catalytic cycle. We found that (a) binding of vinblastine or verapamil alone can cause a conformational change in Pgp, but the change induced by the drug binding is different from that induced by nucleotide binding, (b) there may be at least two binding sites for Pgp substrates, one for drugs such as vinblastine and verapamil and the other for drugs such as colchicine and adriamycin, (c) the conformation of Pgp bound by ATP and vinblastine is different from the conformation bound by either one alone, and (d) the ADP-bound Pgp does not bind vinblastine. Based on these observations and our previous studies, we propose a model for drug binding and transport in the catalytic cycle of Pgp.

Conformational changes of P-glycoprotein by nucleotide binding.

P-glycoprotein (Pgp) is a membrane protein that transports chemotherapeutic drugs, causing multidrug resistance in human cancer cells. Pgp is a member of the ATP-binding cassette superfamily and functions as a transport ATPase. It has been suggested that the conformation of Pgp changes in the catalytic cycle. In this study, we tested this hypothesis by using limited proteolysis as a tool to detect different conformational states trapped by binding of nucleotide ligands and inhibitors. Pgp has high basal ATPase activity; that is, ATP hydrolysis by Pgp is not rigidly associated with drug transport. This activity provides a convenient method for studying the conformational change of Pgp induced by nucleotide ligands, in the absence of drug substrates which may generate complications due to their own binding. Inside-out membrane vesicles containing human Pgp were isolated from multidrug-resistant SKOV/VLB cells and treated with trypsin in the absence or presence of MgATP, Mg-adenosine 5'-[beta,gamma-imido]triphosphate (Mg-p[NH]ppA) and MgADP. Changes in the proteolysis profile of Pgp owing to binding of nucleotides were used to indicate the conformational changes in Pgp. We found that generation of tryptic fragments, including the loop linking transmembrane (TM) regions TM8 and TM9 of Pgp, were stimulated by the binding of Mg-p[NH]ppA, MgATP and MgADP, indicating that the Pgp conformation was changed by the binding of these nucleotides. The effects of nucleotides on Pgp conformation are directly associated with the binding and/or hydrolysis of these ligands. Four conformational states of Pgp were stabilized under different conditions with various ligands and inhibitors. We propose that cycling through these four states couples the Pgp-mediated MgATP hydrolysis to drug transport.