Identification of Methylated Genes Associated with Aggressive Clinicopathological Features in Mantle Cell Lymphoma

Background: Mantle cell lymphoma (MCL) is genetically characterized by the t(11;14)(q13;q32) translocation and a high number of secondary chromosomal alterations. The contribution of DNA methylation to MCL lymphomagenesis is not well known. We sought to identify epigenetically silenced genes in these tumours that might have clinical relevance. Methodology/Principal Findings: To identify potential methylated genes in MCL we initially investigated seven MCL cell lines treated with epigenetic drugs and gene expression microarray profiling. The methylation status of selected candidate genes was validated by a quantitative assay and subsequently analyzed in a series of primary MCL (n=38). After pharmacological reversion we identified 252 potentially methylated genes. The methylation analysis of a subset of these genes (n=25) in the MCL cell lines and normal B lymphocytes confirmed that 80% of them were methylated in the cell lines but not in normal lymphocytes. The subsequent analysis in primary MCL identified five genes (SOX9,HOXA9,AHR,NR2F2 ,and ROBO1) frequently methylated in these tumours. The gene methylation events tended to occur in the same primary neoplasms and correlated with higher proliferation, increased number of chromosomal abnormalities, and shorter survival of the patients. Conclusions: We have identified a set of genes whose methylation degree and gene expression levels correlate with aggressive clinicopathological features of MCL. Our findings also suggest that a subset of MCL might show a CpG island methylator phenotype (CIMP) that may influence the behaviour of the tumours.

The cell cycle–regulated genes of schizosaccharomyces pombe

Many genes are regulated as an innate part of the eukaryotic cell cycle, and a complex transcriptional network helps enable the cyclic behavior of dividing cells. This transcriptional network has been studied in Saccharomyces cerevisiae (budding yeast) and elsewhere. To provide more perspective on these regulatory mechanisms, we have used microarrays to measure gene expression through the cell cycle of Schizosaccharomyces pombe (fission yeast). The 750 genes with the most significant oscillations were identified and analyzed. There were two broad waves of cell cycle transcription, one in early/mid G2 phase, and the other near the G2/M transition. The early/mid G2 wave included many genes involved in ribosome biogenesis, possibly explaining the cell cycle oscillation in protein synthesis in S.pombe. The G2/M wave included at least three distinctly regulated clusters of genes: one large cluster including mitosis, mitotic exit, and cell separation functions, one small cluster dedicated to DNA replication, and another small cluster dedicated to cytokinesis and division. S. pombe cell cycle genes have relatively long, complex promoters containing groups of multiple DNA sequence motifs, often of two, three, or more different kinds. Many of the genes, transcription factors, and regulatory mechanisms are conserved between S. pombe and S. cerevisiae. Finally, we found preliminary evidence for a nearly genome-wide oscillation in gene expression: 2,000 or more genes undergo slight oscillations in expression as a function of the cell cycle, although whether this is adaptive, or incidental to other events in the cell, such as chromatin condensation, we do not know.

Identification of Methylated Genes Associated with Aggressive Clinicopathological Features in Mantle Cell Lymphoma

Background: Mantle cell lymphoma (MCL) is genetically characterized by the t(11;14)(q13;q32) translocation and a high number of secondary chromosomal alterations. The contribution of DNA methylation to MCL lymphomagenesis is not well known. We sought to identify epigenetically silenced genes in these tumours that might have clinical relevance. Methodology/Principal Findings: To identify potential methylated genes in MCL we initially investigated seven MCL cell lines treated with epigenetic drugs and gene expression microarray profiling. The methylation status of selected candidate genes was validated by a quantitative assay and subsequently analyzed in a series of primary MCL (n=38). After pharmacological reversion we identified 252 potentially methylated genes. The methylation analysis of a subset of these genes (n=25) in the MCL cell lines and normal B lymphocytes confirmed that 80% of them were methylated in the cell lines but not in normal lymphocytes. The subsequent analysis in primary MCL identified five genes (SOX9,HOXA9,AHR,NR2F2 ,and ROBO1) frequently methylated in these tumours. The gene methylation events tended to occur in the same primary neoplasms and correlated with higher proliferation, increased number of chromosomal abnormalities, and shorter survival of the patients. Conclusions: We have identified a set of genes whose methylation degree and gene expression levels correlate with aggressive clinicopathological features of MCL. Our findings also suggest that a subset of MCL might show a CpG island methylator phenotype (CIMP) that may influence the behaviour of the tumours.

ROS production is essential for the apoptotic function of E2F1 in pheochromocytoma and neuroblastoma cell lines

In this study we demonstrate that accumulation of reactive oxygen species (ROS) is essential for E2F1 mediated apoptosis in ER-E2F1 PC12 pheochromocytoma, and SH-SY5Y and SK-N-JD neuroblastoma stable cell lines. In these cells, the ER-E2F1 fusion protein is expressed in the cytosol; the addition of 4-hydroxytamoxifen (OHT) induces its translocation to the nucleus and activation of E2F1target genes. Previously we demonstrated that, in ER-E2F1 PC12 cells, OHT treatment induced apoptosis through activation of caspase-3. Here we show that caspase-8 activity did not change upon treatment with OHT. Moreover, over-expression of Bcl-xL arrested OHT-induced apoptosis; by contrast, over-expression of c-FLIP, did not have any effect on OHT-induced apoptosis. OHT addition induces BimL expression, its translocation to mitochondria and activation of Bax, which is paralleled by diminished mitochondrial enrichment of Bcl-xL. Treatment with a Bax-inhibitory peptide reduced OHT-induced apoptosis. These results point out the essential role of mitochondria on the apoptotic process driven by E2F1. ROS accumulation followed E2F1 induction and treatment with the antioxidant N-acetylcysteine, inhibited E2F1-induced Bax translocation to mitochondria and subsequent apoptosis. The role of ROS in mediating OHT-induced apoptosis was also studied in two neuroblastoma cell lines, SH-SY5Y and SK-N-JD. In SH-SY5Y cells, activation of E2F1 by the addition of OHT induced ROS production and apoptosis, whereas over-expression of E2F1 in SK-N-JD cells failed to induce either response. Transcriptional profiling revealed that many of the genes responsible for scavenging ROS were down-regulated following E2F1-induction in SH-SY5Y, but not in SK-N-JD cells. Finally, inhibition of GSK3β blocked ROS production, Bax activation and the down regulation of ROS scavenging genes. These findings provide an explanation for the apparent contradictory role of E2F1 as an apoptotic agent versus a cell cycle activator.

Proteasome inhibition reduces proliferation, collagen expression, and inflammatory cytokine production in nasal mucosa and polyp fibroblasts

Proteasome inhibitors, used in cancer treatment for their proapoptotic effects, have anti-inflammatory and antifibrotic effects on animal models of various inflammatory and fibrotic diseases. Their effects in cells from patients affected by either inflammatory or fibrotic diseases have been poorly investigated. Nasal polyposis is a chronic inflammatory disease of the sinus mucosa characterized by tissue inflammation and remodeling. We tested the hypothesis that proteasome inhibition of nasal polyp fibroblasts might reduce their proliferation and inflammatory and fibrotic response. Accordingly, we investigated the effect of the proteasome inhibitor Z-Leu-Leu-Leu-B(OH)2 (MG262) on cell viability and proliferation and on the production of collagen and inflammatory cytokines in nasal polyp and nasal mucosa fibroblasts obtained from surgery specimens. MG262 reduced the viability of nasal mucosa and polyp fibroblasts concentration- and time-dependently, with marked effects after 48 h of treatment. The proteasome inhibitor bortezomib provoked a similar effect. MG262-induced cell death involved loss of mitochondrial membrane potential, caspase-3 and poly(ADP-ribose) polymerase activation, induction of c-Jun phosphorylation, and mitogen-activated protein kinase phosphatase-1 expression. Low concentrations of MG262 provoked growth arrest, inhibited DNA replication and retinoblastoma phosphorylation, and increased expression of the cell cycle inhibitors p21 and p27. MG262 concentration-dependently inhibited basal and transforming growth factor-β-induced collagen mRNA expression and interleukin (IL)-1β-induced production of IL-6, IL-8, monocyte chemoattractant protein-1, regulated on activation normal T cell expressed and secreted, and granulocyte/macrophage colony-stimulating factor in both fibroblast types. MG262 inhibited IL-1β/tumor necrosis factor-α-induced activation of nuclear factor-κB. We conclude that noncytotoxic treatment with MG262 reduces the proliferative, fibrotic, and inflammatory response of nasal fibroblasts, whereas high MG262 concentrations induce apoptosis.

Proteasome inhibition reduces proliferation, collagen expression, and inflammatory cytokine production in nasal mucosa and polyp fibroblasts

Proteasome inhibitors, used in cancer treatment for their proapoptotic effects, have anti-inflammatory and antifibrotic effects on animal models of various inflammatory and fibrotic diseases. Their effects in cells from patients affected by either inflammatory or fibrotic diseases have been poorly investigated. Nasal polyposis is a chronic inflammatory disease of the sinus mucosa characterized by tissue inflammation and remodeling. We tested the hypothesis that proteasome inhibition of nasal polyp fibroblasts might reduce their proliferation and inflammatory and fibrotic response. Accordingly, we investigated the effect of the proteasome inhibitor Z-Leu-Leu-Leu-B(OH)2 (MG262) on cell viability and proliferation and on the production of collagen and inflammatory cytokines in nasal polyp and nasal mucosa fibroblasts obtained from surgery specimens. MG262 reduced the viability of nasal mucosa and polyp fibroblasts concentration- and time-dependently, with marked effects after 48 h of treatment. The proteasome inhibitor bortezomib provoked a similar effect. MG262-induced cell death involved loss of mitochondrial membrane potential, caspase-3 and poly(ADP-ribose) polymerase activation, induction of c-Jun phosphorylation, and mitogen-activated protein kinase phosphatase-1 expression. Low concentrations of MG262 provoked growth arrest, inhibited DNA replication and retinoblastoma phosphorylation, and increased expression of the cell cycle inhibitors p21 and p27. MG262 concentration-dependently inhibited basal and transforming growth factor-β-induced collagen mRNA expression and interleukin (IL)-1β-induced production of IL-6, IL-8, monocyte chemoattractant protein-1, regulated on activation normal T cell expressed and secreted, and granulocyte/macrophage colony-stimulating factor in both fibroblast types. MG262 inhibited IL-1β/tumor necrosis factor-α-induced activation of nuclear factor-κB. We conclude that noncytotoxic treatment with MG262 reduces the proliferative, fibrotic, and inflammatory response of nasal fibroblasts, whereas high MG262 concentrations induce apoptosis.

CD4 expression decrease by antisense oligonucleotides. Inhibition of rat T CD4+ cell reactivity

In previous studies, we have demonstrated the inhibition of CD4 expression in rat lymphocytes treated with phorbol myristate acetate (PMA) by antisense oligonucleotides (AS-ODNs) directed against the AUG start region of the cd4 gene. The aim of the present study was to inhibit CD4 expression in lymphocytes without promoting CD4 synthesis and to determine the effect of this inhibition on CD4+ T cell function. Four 21-mer ODNs against the rat cd4 gene (AS-CD4-1 to AS-CD4-4) were used. Surface CD4 expression was measured by immunofluorescence staining and flow cytometry, and mRNA CD4 expression was measured by RT-PCR. T CD4+ cell function was determined by specific and unspecific proliferative response of rat-primed lymphocytes. After 24 hours of incubation, AS-CD4-2 and AS-CD4-4 reduced lymphocyte surface CD4 expression by 40%. This effect remained for 72 hours and was not observed on other surface molecules, such as CD3, CD5, or CD8. CD4 mRNA expression was reduced up to 40% at 24 hours with AS-CD4-2 and AS-CD4-4. After 48 hours treatment, CD4 mRNA decreased up to 27% and 29% for AS-CD4-2 and AS-CD4-4, respectively. AS-CD4-2 and AS-CD4-4 inhibited T CD4+ cell proliferative response upon antigen-specific and unspecific stimuli. Therefore, AS-ODNs against CD4 molecules inhibited surface and mRNA CD4 expression, under physiologic turnover and, consequently, modulate T CD4+ cell reactivity.

CD4 expression decrease by antisense oligonucleotides. Inhibition of rat T CD4+ cell reactivity

In previous studies, we have demonstrated the inhibition of CD4 expression in rat lymphocytes treated with phorbol myristate acetate (PMA) by antisense oligonucleotides (AS-ODNs) directed against the AUG start region of the cd4 gene. The aim of the present study was to inhibit CD4 expression in lymphocytes without promoting CD4 synthesis and to determine the effect of this inhibition on CD4+ T cell function. Four 21-mer ODNs against the rat cd4 gene (AS-CD4-1 to AS-CD4-4) were used. Surface CD4 expression was measured by immunofluorescence staining and flow cytometry, and mRNA CD4 expression was measured by RT-PCR. T CD4+ cell function was determined by specific and unspecific proliferative response of rat-primed lymphocytes. After 24 hours of incubation, AS-CD4-2 and AS-CD4-4 reduced lymphocyte surface CD4 expression by 40%. This effect remained for 72 hours and was not observed on other surface molecules, such as CD3, CD5, or CD8. CD4 mRNA expression was reduced up to 40% at 24 hours with AS-CD4-2 and AS-CD4-4. After 48 hours treatment, CD4 mRNA decreased up to 27% and 29% for AS-CD4-2 and AS-CD4-4, respectively. AS-CD4-2 and AS-CD4-4 inhibited T CD4+ cell proliferative response upon antigen-specific and unspecific stimuli. Therefore, AS-ODNs against CD4 molecules inhibited surface and mRNA CD4 expression, under physiologic turnover and, consequently, modulate T CD4+ cell reactivity.

Progressive Purkinje Cell Degeneration in tambaleante Mutant Mice Is a Consequence of a Missense Mutation in HERC1 E3 Ubiquitin Ligase

The HERC gene family encodes proteins with two characteristic domains: HECT and RCC1-like. Proteins with HECT domain shave been described to function as ubiquitin ligases, and those that contain RCC1-like domains have been reported to function as GTPases regulators. These two activities are essential in a number of important cellular processes such as cell cycle, cell signaling, and membrane trafficking. Mutations affecting these domains have been found associated with retinitis pigmentosa, amyotrophic lateral sclerosis, and cancer. In humans, six HERC genes have been reported which encode two subgroups of HERC proteins: large (HERC1-2) and small (HERC3-6). The giant HERC1 protein was the first to be identified. It has been involved in membrane trafficking and cell proliferation/growth through its interactions with clathrin, M2-pyruvate kinase, and TSC2 proteins. Mutations affecting other members of the HERC family have been found to be associated with sterility and growth retardation. Here, we report the characterization of a recessive mutation named tambaleante, which causes progressive Purkinje cell degeneration leading to severe ataxia with reduced growth and lifespan in homozygous mice aged over two months. We mapped this mutation in mouse chromosome 9 and then performed positional cloning. We found a GuA transition at position 1448, causing a Gly to Glu substitution (Gly483Glu) in the highly conserved N- terminal RCC1-like domain of the HERC1 protein. Successful transgenic rescue, with either a mouse BAC containing the normal copy of Herc1 or with the human HERC1 cDNA, validated our findings. Histological and biochemical studies revealed extensive autophagy associated with an increase of the mutant protein level and a decrease of mTOR activity. Our observations concerning this first mutation in the Herc1 gene contribute to the functional annotation of the encoded E3 ubiquitin ligase and underline the crucial and unexpected role of this protein in Purkinje cell physiology.

Utilització de plantes d'Arabidopsis thaliana mutants de CK2 per a l'establiment de línies cel·lulars i per a l'estudi de la implicació de la CK2 en l'estructuració de la cromatina

La proteïna CK2 és una Ser/Thr fosfotransferasa evolutivament conservada. Està formada per dues subunitats diferents, α (catalítica) i β (reguladora), que s’associen en un complex heterotetramèric (α2β2). L’activitat d’aquest enzim està regulada per varis mecanismes que la modulen diferencialment en funció del substrat, entre ells, la localització subcel·lular de les diferents subunitats. A partir de plantes transgèniques que contenen les subunitats de CK2 fusionades a una proteïna fluorescent (GFP o YFP) hem establert línies cel·lulars que permetran analitzar, en treballs futurs, els canvis de localització subcel·lular de les subunitats de CK2 provocats per diferents estímuls. La CK2 intervé en un gran nombre de processos cel·lulars, entre d’altres, metabolisme, transport a nucli, control del cicle cel·lular i reparació del DNA. Amb la finalitat d’estudiar el paper de la CK2 en el desenvolupament de les plantes, membres del grup van generar un mutant dominant negatiu per transformació estable de plantes d’Arabidopsis thaliana. Estudis previs mostraven que la pèrdua d’activitat CK2 en aquestes plantes provocava una alteració de l’activitat mitòtica i un bloqueig del cicle cel·lular. Els resultats obtinguts en aquest treball indiquen que aquests fenotips podrien ser conseqüència de la implicació de la CK2 en processos d’estructuració de la cromatina.