Caspase-activated DNase is necessary and sufficient for oligonucleosomal DNA breakdown, but not for chromatin disassembly during caspase-dependent apoptosis of LN-18 glioblastoma cells.

Caspase-dependent apoptosis is a controlled type of cell death characterized by oligonucleosomal DNA breakdown and major nuclear morphological alterations. Other kinds of cell death do not share these highly distinctive traits because caspase-activated DNase (DFF40/CAD) remains inactive. Here, we report that human glioblastoma multiforme-derived LN-18 cells do not hydrolyze DNA into oligonucleosomal fragments after apoptotic insult. Furthermore, their chromatin remains packaged into a single mass, with no signs of nuclear fragmentation. However, ultrastructural analysis reveals that nuclear disassembly occurs, although compacted chromatin does not localize into apoptotic nuclear bodies. Caspases become properly activated, and ICAD, the inhibitor of DFF40/CAD, is correctly processed. Using cell-free in vitro assays, we show that chromatin from isolated nuclei of LN-18 cells is suitable for hydrolysis into oligonuclesomal fragments by staurosporine-pretreated SH-SY5Y cytoplasms. However, staurosporine-pretreated LN-18 cytoplasms do not induce DNA laddering in isolated nuclei from either LN-18 or SH-SY5Y cells because LN-18 cells express lower amounts of DFF40/CAD. DFF40/CAD overexpression makes LN-18 cells fully competent to degrade their DNA into oligonucleosome-sized fragments, and yet they remain unable to arrange their chromatin into nuclear clumps after apoptotic insult. Indeed, isolated nuclei from LN-18 cells were resistant to undergoing apoptotic nuclear morphology in vitro. The use of LN-18 cells has uncovered a previously unsuspected cellular model, whereby a caspase-dependent chromatin package is DFF40/CAD-independent, and DFF40/CAD-mediated double-strand DNA fragmentation does not warrant the distribution of the chromatin into apoptotic nuclear bodies. The studies highlight a not-yet reported DFF40/CAD-independent mechanism driving conformational nuclear changes during caspase-dependent cell death.

Smad2 and Smad3 cooperate and antagonize simultaneously in vertebrate neurogenesis.

The transforming growth factor beta (TGF-ß) pathway plays key roles in development and cancer. TGF-ß signaling converges on the Smad2 and Smad3 effectors, which can either cooperate or antagonize to regulate their transcriptional targets. Here we performed in vivo and in silico experiments to study how such cooperativity and antagonism might function during neurogenesis. In vivo electroporation experiments in the chick embryo neural tube show that Smad2 and Smad3 cooperate to promote neurogenesis, as well as the transcription of Smad3-specific targets. Knockdown of Smad2 enhances neurogenesis and the transcription of Smad3-specific targets. A mathematical model of the TGF-ß pathway fits the experimental results and predicts that the proportions of the three different trimeric complexes formed dictates the transcriptional responses of the R-Smad proteins. As such, Smad2 targets are activated solely by the Smad2-Smad2-Smad4 complex, whereas Smad3 targets are activated both by Smad2-Smad3-Smad4 and Smad3-Smad3-Smad4 trimers. We have modeled the Smad responses onto arbitrary genes and propose that this mechanism might be extended to additional activities of TGF-ß in development and disease.

LMO4 is an essential cofactor in the Snail2-mediated epithelial-to-mesenchymal transition of neuroblastoma and neural crest cells.

Neuroblastoma is an embryonic tumor derived from cells of the neural crest. Taking advantage of a newly developed neural crest lineage tracer and based on the hypothesis that the molecular mechanisms that mediate neural crest delamination are also likely to be involved in the spread of neuroblastoma, we were able to identify genes that are active both in neural crest development and neuroblastoma tumor formation. A subsequent search of the neuroblastoma gene server for human orthologues of genes differentially expressed in the chick embryo neural crest screen retrieved the LIM domain only protein 4 (LMO4), which was expressed in both cell types analyzed. Functional experiments in these two model systems revealed that LMO4 activity is required for neuroblastoma cell invasion and neural crest delamination. Moreover, we identified LMO4 as an essential cofactor in Snail2-mediated cadherin repression and in the epithelial-to-mesenchymal transition of both neural crest and neuroblastoma cells. Together, our results suggest that the association of high levels of LMO4 with aggressive neuroblastomas is dependent on LMO4 regulation of cadherin expression and hence, tumor invasiveness.

Chromatin collapse during caspase-dependent apoptotic cell death requires DNA fragmentation factor, 40-kDa subunit-/caspase-activated deoxyribonuclease-mediated 3'-OH single-strand DNA breaks.

Apoptotic nuclear morphology and oligonucleosomal double-strand DNA fragments (also known as DNA ladder) are considered the hallmarks of apoptotic cell death. From a classic point of view, these two processes occur concomitantly. Once activated, DNA fragmentation factor, 40-kDa subunit (DFF40)/caspase-activated DNase (CAD) endonuclease hydrolyzes the DNA into oligonucleosomal-size pieces, facilitating the chromatin package. However, the dogma that the apoptotic nuclear morphology depends on DNA fragmentation has been questioned. Here, we use different cellular models, including MEF CAD(-/-) cells, to unravel the mechanism by which DFF40/CAD influences chromatin condensation and nuclear collapse during apoptosis. Upon apoptotic insult, SK-N-AS cells display caspase-dependent apoptotic nuclear alterations in the absence of internucleosomal DNA degradation. The overexpression of a wild-type form of DFF40/CAD endonuclease, but not of different catalytic-null mutants, restores the cellular ability to degrade the chromatin into oligonucleosomal-length fragments. We show that apoptotic nuclear collapse requires a 3'-OH endonucleolytic activity even though the internucleosomal DNA degradation is impaired. Moreover, alkaline unwinding electrophoresis and In Situ End-Labeling (ISEL)/In Situ Nick Translation (ISNT) assays reveal that the apoptotic DNA damage observed in the DNA ladder-deficient SK-N-AS cells is characterized by the presence of single-strand nicks/breaks. Apoptotic single-strand breaks can be impaired by DFF40/CAD knockdown, abrogating nuclear collapse and disassembly. In conclusion, the highest order of chromatin compaction observed in the later steps of caspase-dependent apoptosis relies on DFF40/CAD-mediated DNA damage by generating 3'-OH ends in single-strand rather than double-strand DNA nicks/breaks.

The calcium-sensing receptor is silenced by genetic and epigenetic mechanisms in unfavorable neuroblastomas and its reactivation induces ERK1/2-dependent apoptosis.

Neuroblastic tumors (NTs) include the neuroblastomas, ganglioneuroblastomas and ganglioneuromas. We have reported previously that the calcium-sensing receptor is expressed in differentiated, favorable NTs but almost undetectable in unfavorable neuroblastomas. We have now detected hypermethylation of a particular region within the CpG island encompassing the CaSR gene promoter 2 in neuroblastoma cell lines and 25% primary neuroblastomas. Hypermethylation of this region was associated with reduced CaSR messenger RNA expression and several predictors of poor outcome in neuroblastomas, including MYCN amplification. Treatment with 5'aza-2-deoxycitidine and/or trichostatin A restored CaSR expression in MYCN-amplified cell lines. Following 5'aza-2-deoxycitidine exposure, decreased percentages of methylated CpG sites were observed at the above-mentioned region. By interphase fluorescence in situ hybridization, variable percentages of nuclei with monosomy of chromosome 3, where the human CaSR gene resides, were observed in more than 90% of primary NTs of all subgroups. Nuclei harboring this alteration were heterogeneously distributed among tumor cells. Ectopic overexpression of the calcium-sensing receptor in two MYCN-amplified neuroblastoma cell lines in which this gene is silenced by promoter hypermethylation significantly reduced their in vitro proliferation rates and almost abolished their capacity to generate xenografts in immunocompromised mice. Finally, upon acute exposure to calcium, the primary activator of this receptor, calcium-sensing receptor-overexpressing neuroblastoma cells underwent apoptosis, a process dependent on sustained activation of ERK1/2. These data would support the hypothesis that epigenetic silencing of the CaSR gene is neither an in vitro artefact in neuroblastoma cell lines nor an irrelevant, secondary event in primary NTs, but a significant mechanism for neuroblastoma survival.

Apoptotic DNA degradation into oligonucleosomal fragments, but not apoptotic nuclear morphology, relies on a cytosolic pool of DFF40/CAD endonuclease.

Apoptotic cell death is characterized by nuclear fragmentation and oligonucleosomal DNA degradation, mediated by the caspase-dependent specific activation of DFF40/CAD endonuclease. Here, we describe how, upon apoptotic stimuli, SK-N-AS human neuroblastoma-derived cells show apoptotic nuclear morphology without displaying concomitant internucleosomal DNA fragmentation. Cytotoxicity afforded after staurosporine treatment is comparable with that obtained in SH-SY5Y cells, which exhibit a complete apoptotic phenotype. SK-N-AS cell death is a caspase-dependent process that can be impaired by the pan-caspase inhibitor q-VD-OPh. The endogenous inhibitor of DFF40/CAD, ICAD, is correctly processed, and dff40/cad cDNA sequence does not reveal mutations altering its amino acid composition. Biochemical approaches show that both SH-SY5Y and SK-N-AS resting cells express comparable levels of DFF40/CAD. However, the endonuclease is poorly expressed in the cytosolic fraction of healthy SK-N-AS cells. Despite this differential subcellular distribution of DFF40/CAD, we find no differences in the subcellular localization of both pro-caspase-3 and ICAD between the analyzed cell lines. After staurosporine treatment, the preferential processing of ICAD in the cytosolic fraction allows the translocation of DFF40/CAD from this fraction to a chromatin-enriched one. Therefore, the low levels of cytosolic DFF40/CAD detected in SK-N-AS cells determine the absence of DNA laddering after staurosporine treatment. In these cells DFF40/CAD cytosolic levels can be restored by the overexpression of their own endonuclease, which is sufficient to make them proficient at degrading their chromatin into oligonucleosome-size fragments after staurosporine treatment. Altogether, the cytosolic levels of DFF40/CAD are determinants in achieving a complete apoptotic phenotype, including oligonucleosomal DNA degradation.