Nucleophagy contributes to genome stability through degradation of type II topoisomerases A and B and nucleolar components.

The nuclear architecture of mammalian cells can be altered as a consequence of anomalous accumulation of nuclear proteins or genomic alterations. Most of the knowledge about nuclear dynamics comes from studies on cancerous cells. How normal healthy cells maintain genome stability, avoiding accumulation of nuclear damaged material, is less understood. Here, we describe that primary mouse embryonic fibroblasts develop a basal level of nuclear buds and micronuclei, which increase after etoposide-induced DNA double-stranded breaks. Both basal and induced nuclear buds and micronuclei colocalize with the autophagic proteins BECN1 and LC3B (also known as MAP1LC3B) and with acidic vesicles, suggesting their clearance by nucleophagy. Some of the nuclear alterations also contain autophagic proteins and type II DNA topoisomerases (TOP2A and TOP2B), or the nucleolar protein fibrillarin, implying they are also targets of nucleophagy. We propose that basal nucleophagy contributes to genome and nuclear stability, as well as in response to DNA damage.

Role of autophagy in the chemopreventive effect of the IFC-305 compound in the sequential model of cirrhosis-hepatocellular carcinoma in the rat and in vitro.

Hepatocellular carcinoma (HCC) can be originated from various etiologies and is preceded mostly by cirrhosis. Unfortunately, there is no effective treatment due to its late prognosis. Alterations in autophagy have been reported during the development and progression of HCC. Autophagy allows for the maintenance of a positive energy balance and the proper functioning of organelles through the selective degradation of cellular components. It has been demonstrated that autophagy suppresses spontaneous tumorigenesis in the liver. Therefore, autophagy has become a therapeutic target for effective HCC therapies. We have previously demonstrated that the adenosine-derived compound, IFC-305, has a chemopreventive effect on HCC, in addition to maintaining mitochondrial function in a sequential model of cirrhosis-HCC. Thus, the aim of this work was to determine if IFC-305 has an effect on autophagy in the sequential model of cirrhosis-HCC induced by diethylnitrosamine or in vitro in the HCC cell line HepG2 and mouse embryonic fibroblasts. The results of this work showed that IFC-305 modifies the levels of the BECN1, p62/SQSTM1 and LC3-II proteins that play an important role in the autophagic process. In vivo, IFC-305 regulates the levels of the PINK1 and PARKIN proteins that specifically mark mitochondria for repair or degradation. In the HepG2 cell line, its effect was accompanied by a decrease in cell viability. Interestingly, in nontumoral cells the time to autophagy induction was different compared to the HepG2 cells. This study suggests that autophagy induction may be part of the mechanism by which IFC-305 maintains mitochondrial function, thereby facilitating the prevention and reversal of HCC.

The nuclear receptor NR4A1 is regulated by SUMO modification to induce autophagic cell death.

NR4A is a nuclear receptor protein family whose members act as sensors of cellular environment and regulate multiple processes such as metabolism, proliferation, migration, apoptosis, and autophagy. Since the ligand binding domains of these receptors have no cavity for ligand interaction, their function is most likely regulated by protein abundance and post-translational modifications. In particular, NR4A1 is regulated by protein abundance, phosphorylation, and subcellular distribution (nuclear-cytoplasmic translocation), and acts both as a transcription factor and as a regulator of other interacting proteins. SUMOylation is a post-translational modification that can affect protein stability, transcriptional activity, alter protein-protein interactions and modify intracellular localization of target proteins. In the present study we evaluated the role of SUMOylation as a posttranslational modification that can regulate the activity of NR4A1 to induce autophagy-dependent cell death. We focused on a model potentially relevant for neuronal cell death and demonstrated that NR4A1 needs to be SUMOylated to induce autophagic cell death. We observed that a triple mutant in SUMOylation sites has reduced SUMOylation, increased transcriptional activity, altered intracellular distribution, and more importantly, its ability to induce autophagic cell death is impaired.

Cortical neurons develop a senescence-like phenotype promoted by dysfunctional autophagy.

Senescent cells accumulate in various tissues and organs with aging altering surrounding tissue due to an active secretome, and at least in mice their elimination extends healthy lifespan and ameliorates several chronic diseases. Whether all cell types senesce, including post-mitotic cells, has been poorly described mainly because cellular senescence was defined as a permanent cell cycle arrest. Nevertheless, neurons with features of senescence have been described in old rodent and human brains. In this study we characterized an in vitro model useful to study the molecular basis of senescence of primary rat cortical cells that recapitulates senescent features described in brain aging. We found that in long-term cultures, rat primary cortical neurons displayed features of cellular senescence before glial cells did, and developed a functional senescence-associated secretory phenotype able to induce paracrine premature senescence of mouse embryonic fibroblasts but proliferation of rat glial cells. Functional autophagy seems to prevent neuronal senescence, as we observed an autophagic flux reduction in senescent neurons both in vitro and in vivo, and autophagy impairment induced cortical cell senescence while autophagy stimulation inhibited it. Our findings suggest that aging-associated dysfunctional autophagy contributes to senescence transition also in neuronal cells.

Prevalence and Clinical Significance of FLT3 Mutation Status in Acute Myeloid Leukemia Patients: A Multicenter Study.

BACKGROUND AND AIMS: FLT3-ITD mutations in acute myeloid leukemia (AML) are associated with a poor prognosis. In Latin America, little epidemiological data exist about these mutations and their influence on clinical evolution and prognosis. Standardization and well-established clinical correlation make FLT3 mutational analysis by molecular methods an invaluable tool to decide among treatment options and to determine AML prognosis. METHODS: We assessed the prevalence of FLT3-ITD mutations in 138 patients with AML at four hematology referral centers from Mexico and Colombia. Molecular methods based on polymerase chain reaction (PCR) were employed for determining FLT3-ITD status. RESULTS: Mutations were present in 28 patients indicating a prevalence of 20.28%. Median age was 47 years (5-96). The FLT3 mutation positive group was older, had higher WBC and hemoglobin values and lower platelet counts but without statistical significance. A not previously described mutation in the FLT3 gene was found in one patient involving a nucleotide exchange of timine for cytosine at the 66608 position. A high mortality was found in the FLT3-mutated group, 67.8 vs. 42.72% in the non-mutated group and median survival was 4.9 months vs. 20.4 months, p = 0.009. A mutated FLT3 did not confer poor prognosis to those with M3 AML. The mutated FLT3 population had poor overall survival (OS) despite hematoprogenitor stem cell transplantation (HSCT). CONCLUSION: Prevalence of FLT3-ITD mutation in AML was present in a proportion comparable to other populations and, when present, was associated with a very poor prognosis.