Blockade of the NFκB pathway drives differentiating glioblastoma-initiating cells into senescence both in vitro and in vivo.

Glioblastoma multiforme is one of the most devastating cancers and presents unique challenges to therapy because of its aggressive behavior. Cancer-initiating or progenitor cells have been described to be the only cell population with tumorigenic capacity in glioblastoma. Therefore, effective therapeutic strategies targeting these cells or the early precursors may be beneficial. We have established different cultures of glioblastoma-initiating cells (GICs) derived from surgical specimens and found that, after induction of differentiation, the NFκB transcriptional pathway was activated, as determined by analyzing key proteins such as p65 and IκB and the upregulation of a number of target genes. We also showed that blockade of nuclear factor (NF)κB signaling in differentiating GICs by different genetic strategies or treatment with small-molecule inhibitors, promoted replication arrest and senescence. This effect was partly mediated by reduced levels of the NFκB target gene cyclin D1, because its downregulation by RNA interference reproduced a similar phenotype. Furthermore, these results were confirmed in a xenograft model. Intravenous treatment of immunodeficient mice bearing human GIC-derived tumors with a novel small-molecule inhibitor of the NFκB pathway induced senescence of tumor cells but no ultrastructural alterations of the brain parenchyma were detected. These findings reveal that activation of NFκB may keep differentiating GICs from acquiring a mature postmitotic phenotype, thus allowing cell proliferation, and support the rationale for therapeutic strategies aimed to promote premature senescence of differentiating GICs by blocking key factors within the NFκB pathway.

SUMO-1 transiently localizes to Cajal bodies in mammalian neurons.

Cajal bodies (CBs) are nuclear organelles involved in the maturation of small nuclear ribonucleoproteins required for the processing of pre-mRNAs. They concentrate coilin, splicing factors and the survival of motor neuron protein (SMN). By using immunocytochemistry and transfection experiments with GFP-SUMO-1, DsRed1-Ubc9, GFP-coilin and GFP-SMN constructs we demonstrate the presence of SUMO-1 and the SUMO conjugating enzyme (Ubc9) in a subset of CBs in undifferentiated neuron-like UR61 cells. Furthermore, SUMO-1 is transiently localized into neuronal CBs from adult nervous tissue in response to osmotic stress or inhibition of methyltransferase activity. SUMO-1-positive CBs contain coilin, SMN and small nuclear ribonucleoproteins, suggesting that they are functional CBs involved in pre-mRNA processing. Since coilin and SMN have several putative motifs of SUMO-1 modification, we suggest that the sumoylation of coilin and/or SMN might play a role in the molecular reorganization of CBs during the neuronal differentiation or stress-response.

Nuclear organization and dynamics of transcription sites in rat sensory ganglia neurons detected by incorporation of 5'-fluorouridine into nascent RNA.

In this study we have used the transcription assay with 5'-fluorouridine incorporation into nascent RNA to analyze the nuclear organization and dynamics of transcription sites in rat trigeminal ganglia neurons. The 5'-FU administrated by i.p. injection was successfully incorporated into nuclear domains containing actively transcribing genes of trigeminal neurons. 5'-Fluorouridine RNA-labeling was detected with immunocytochemistry at light and electron microscopy levels. The 5'-fluorouridine incorporation sites were detected in the nucleolus, particularly on the dense fibrillar component, and in numerous transcription foci spread throughout the euchromatin regions, without preferential positioning at the nuclear periphery or in the nuclear interior. Double labeling experiments to combine 5'-fluorouridine incorporation with molecular markers of nuclear compartments showed the absence of transcription sites in Cajal bodies and nuclear speckles of splicing factors. Similarly, no 5'-fluorouridine labeling was detected in well-characterized chromatin silencing domain, the telomeric heterochromatin. The specificity and sensitivity of the run-on transcription assay in trigeminal ganglia neurons was verified by the i.p. administration of the transcription inhibitor actinomycin D. The dramatic reduction in RNA synthesis upon actinomycin D treatment was associated with two important cellular events, heterochromatin silencing and formation of DNA damage/repair nuclear foci, demonstrated by the expression of tri-methylated histone H4 and phosphorylated H2AX, respectively. 5'-Fluorouridine incorporation in animal models provides a useful tool to investigate the organization of gene expression in mammalian neurons in both normal physiology and experimental pathology systems.

Reorganization of nuclear compartments of type A neurons of trigeminal ganglia in response to inflammatory injury of peripheral nerve endings.

In this study we have taken advantage of the high nuclear responsiveness of type A sensory ganglia neurons to variations of cellular activity to investigate the reorganization and dynamics of nuclear compartments involved in transcription and RNA processing in response to neuronal injury. As experimental model we have used the inflammatory injury of the peripheral nerve endings induced by formalin injection in the areas of ophthalmic/maxillary nerve distribution. We have performed immunofluorescence and confocal laser microscopy analysis with specific antibodies for different nuclear compartments and ultrastructural analysis. The initial response to neuronal injury, within the 3 days post-injury, consisted of chromatin condensation, reduction in the expression level of acetylated histone H4, accumulation of perichromatin granules, reorganization of splicing factors in prominent nuclear speckles, reduction in the number of Cajal bodies and nucleolar alterations. These changes tended to revert by day 7 post-injury and are consistent with a transient inhibition of transcription and RNA processing. Moreover, we have observed an early and sustained expression of the transcription factor c-Jun. These results illustrate the transcription-dependent organization of nuclear compartments in type A trigeminal neurons and also support the importance of the nuclear response to axonal injury as a key component in the regenerative capacity of this neuronal population.