To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES) cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP). A total of 2%–4% of the cells generated a functional GFP by homology-directed repair (HR) and gene conversion. However, ~50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2′-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments.

Dual-specificity phosphatase 6 (DUSP6, mitogen-activated protein kinase (MAPK) phosphatase 3 or PYST1) dephosphorylates phosphotyrosine and phosphothreonine residues on extracellular signal-regulated kinase (ERK1/ 2; MAPK1/2) to inactivate the ERK1/2 kinase. DUSP6 is a critical regulator of the ERK signaling cascade and has been implicated as a tumor suppressor. We report here experimental evidences that DUSP6 is transcriptionally upregulated in primary and long-term cultures of human glioblastoma, as assayed by northern hybridization and real-time quantitative PCR, producing constitutive high level of protein expression. Functional assays were performed with adenovirus-mediated expression of DUSP6 in glioblastoma cultures. Protein overexpression inhibits growth by inducing G1-phase delay and increased mitogenic/anchorage dependence and clonogenic potential in vitro. Changes in cell morphology were associated with an increased tumor growth in vivo. Chemoresistance is a major cause of treatment failure and poor outcome in human glioblastomas. Importantly, DUSP6 overexpression increased resistance to cisplatinmediated cell death in vitro and in vivo. Antisense-mediated depletion of DUSP6 acted in lowering the threshold to anticancer DNA-damaging drugs. We conclude that upregulation of DUSP6 exerts a tumor-promoting role in human glioblastomas exacerbating the malignant phenotype.

We describe the use of phage libraries to derive new antibodies against p21Ras to be used for intracellular expression in mammalian cells. A panel of single-chain antibody fragments, binding to Ras, were analyzed and characterized for their capacity to interfere in vitro with (a) the intrinsic GTPase activity of Ras and (b) the binding of Ras to its effector Raf, and were found not to neutralize its function, according to these biochemical criteria. When expressed intracellularly in mouse 3T3 K-Ras transformed cells all the anti-Ras single-chain variable fragments (scFv) tested inhibited cell proliferation, as assessed by bromodeoxyuridine incorporation. Double immunofluorescence analysis of transfected cells using confocal microscopy confirmed that anti-Ras antibody fragments colocalize with endogenous Ras, at subcellular locations where the protein Ras is not normally found. These data suggest that the ability of phage-derived anti-Ras scFv fragments to inhibit the function of Ras in vivo is a rather general and frequent property and that the range of antibodies that can be successfully used for intracellular inhibition studies is much greater than anticipated, exploiting the mode of action of diverting protein traffic.Keywords: intracellular antibodies; scFv fragments; anti-p21Ras; phage display library.The antibody-based intracellular immunization strategy exploits the ectopic expression of recombinant antibodies to redirect antibodies to different intracellular compartments, to inhibit the function of selected antigens in different biological systems [1±3]. By using this approach the function of several intracellular gene products has been successfully inhibited in the cytoplasm, in the nucleus and in the secretory compartments [4±9]. Intracellular antibodies have been prospected as tools for gene therapy [10] and for functional genomics [11]. In a gene therapy setting, the therapeutic antibody is likely to be the outcome of a long and careful optimization process. On the other hand, in the context of functional genomics project, where intracellular antibodies could be used in a high throughput fashion to assess the function of genes coming from genome projects, the development of reliable methods linking the isolation of recombinant antibodies derived from phage libraries to their intracellular expression in mammalian cells is required. This strategy would require that a high proportion of the isolated antibodies are functional when expressed as intrabodies in the cell cytoplasm.The aim of our paper was to investigate what proportion of the antibody fragments isolated from phage libraries are effective as intrabodies and to assess whether in vivo inhibition of protein function is crucially dependent on a particular antibody with well defined properties or whether it can be obtained by a wide spectrum of single-chain variable fragments (scFv). To this aim we exploited the Ras system, a signal transduction protein which has been successfully inhibited with one intracellular antibody, derived from the well ch...

Phosphatidylinositol 3-kinase (PI3K) is necessary for thyroid stimulating hormone (TSH)-induced cell cycle progression. To determine the molecular mechanism linking PI3K to TSH, we have identified a serine residue in p85a PI3K phosphorylated by protein kinase A (PKA) in vitro and in vivo. Expression of an alanine mutant (p85A) abolished cyclic AMP/TSH-induced cell cycle progression and was lethal in thyroid cells . The aspartic version of the p85a PI3K (p85D) inhibited apoptosis following TSH withdrawal. The p85a PI3K wild type not the p85A bound PKA regulatory subunit RIIb in cells stimulated with cAMP or TSH. The binding of the aspartic version of p85a PI3K to RIIb was independent of cAMP or TSH stimulation. Similarly, binding of PI3K to p21Ras and activation of AKT, a downstream PI3K target, were severely impaired in cells expressing the p85A mutant. Finally, we found that the catalytic activity of PI3K was stimulated by TSH in cells expressing the wildtype p85a PI3K but not in cells expressing p85A. This latter mutant did not affect the epidermal growth factorstimulated PI3K activity. We suggest that (1) TSHcAMP-induced PKA phosphorylates p85a PI3K at serine 83, (2) phosphorylated p85a PI3K binds RIIb-PKA and targets PKAII to the membrane, and (3) PI3K activity and p21Ras binding to PI3K increase and activate PI3K downstream targets. This pathway is essential for the transmission of TSH-cAMP growth signals.

Acute myeloid leukemia (AML) is a heterogeneous hematopoietic malignancy characterized by the accumulation of incompletely differentiated progenitor cells (blasts) in the bone marrow and blood, and by suppression of normal hematopoiesis. It has recently become apparent that the AML genome is characterized by recurrent mutations and dysregulations in epigenetic regulators. These mutations frequently occur before the onset of full blown leukemia, at the pre-leukemic phase, and persist in residual disease that remains after therapeutic intervention, thus suggesting that targeting the AML epigenome may help to eradicate minimal residual disease and prevent relapse. Within the AML epigenome, lysine-specific demethylase 1 A (LSD1) is a histone demethylase that is found frequently overexpressed, albeit not mutated, in AML. LSD1 is a required constituent of critical transcription repressor complexes like CoREST and nucleosome remodeling and deacetylase (NuRD), and abrogation of LSD1 expression results in impaired self-renewal and proliferation, and increased differentiation and apoptosis in AML models and primary cells, particularly in AMLs with MLL- and AML1-rearrangements, or erythroid and megakaryoblastic differentiation block. On this basis, a number of LSD1 inhibitors have been developed in the past decade, and few of them are currently being tested in clinical trials for patients with AML, along with other malignancies. To date, the most promising application of this therapeutic strategy appears to be combination therapy of LSD1 inhibitors with all-trans retinoic acid (ATRA) to reactivate myeloid differentiation in cells that are not spontaneously susceptible to ATRA treatment. In this review, we provide an overview of LSD1 function in normal hematopoiesis and leukemia, and of the current clinical application of LSD1 inhibitors for the treatment of patients with AML.

The anti-p21ras Y13-259 single-chain Fv fragment (scFv) neutralizes the activity of p21-ras when intracellularly expressed in different systems. We have studied the mode of action of this inhibition in 3T3 K-ras fibroblasts and demonstrated that (i) this antibody fragment is highly aggregating when cytoplasmically expressed and (ii) the p21-ras antigen is sequestered in these aggregates in an antibody-dependent manner. This co-segregation leads to an efficient inhibition of DNA synthesis. These results suggest that an antigen can be diverted from its normal location inside the cells in an antibody mediated way, prospecting a new mode of action for intracellular antibodies in vivo.z 1998 Federation of European Biochemical Societies.

To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES) cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP). A total of 2%-4% of the cells generated a functional GFP by homology-directed repair (HR) and gene conversion. However, ;50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-29-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments.Citation: Cuozzo C, Porcellini A, Angrisano T, Morano A, Lee B, et al. (2007) DNA damage, homology-directed repair, and DNA methylation. PLoS Genet 3(7): e110.

Reactive oxygen and nitrogen species (ROS and RNS, respectively) activate the redox-sensitive Ras small GTPases. The three canonical genes ( HRAS, NRAS, and KRAS ) are archetypes of the superfamily of small GTPases and are the most common oncogenes in human cancer. Oncogenic Ras is intimately linked to redox biology, mainly in the context of tumorigenesis. The Ras protein structure is highly conserved, especially in effector-binding regions. Ras small GTPases are redox-sensitive proteins thanks to the presence of the NKCD motif (Asn116-Lys 117-Cys118-Asp119). Notably, the ROS- and RNS-based oxidation of Cys118 affects protein stability, activity, and localization, and protein-protein interactions. Cys residues at positions 80, 181, 184, and 186 may also help modulate these actions. Moreover, oncogenic mutations of Gly12Cys and Gly13Cys may introduce additional oxidative centres and represent actionable drug targets. Here, the pathophysiological involvement of Cys-redox regulation of Ras proteins is reviewed in the context of cancer and heart and brain diseases.