Histone demethylase JMJD3 at the intersection of cellular senescence and cancer.

Cellular senescence is defined by an irreversible growth arrest and is an important biological mechanism for suppression of tumor formation. Although deletion/mutation to DNA sequences is one mechanism by which cancer cells can escape senescence, little is known about the epigenetic factors contributing to this process. Histone modifications and chromatin remodeling related to the function of a histone demethylase, jumonji domain-containing protein 3 (JMJD3; also known as KDM6B), play an important role in development, tissue regeneration, stem cells, inflammation, and cellular senescence and aging. The role of JMJD3 in cancer is poorly understood and its function may be at the intersection of many pathways promoted in a dysfunctional manner such as activation of the senescence-associated secretory phenotype (SASP) observed in aging.

Cellular uptake and retention studies of silica nanoparticles utilizing senescent fibroblasts.

Understanding the interplay between nanoparticles (NPs) and cells is essential to designing more efficient nanomedicines. Previous research has shown the role of the cell cycle having impact on the efficiency of cellular uptake and accumulation of NPs. However, there is a limited investigation into the biological fate of NPs in cells that are permanently withdrawn from the cell cycle. Here we utilize senescent WI-38 fibroblasts, which do not divide and provide a definitive model for tracking the biological fate of silica nanoparticles (SiNPs) independent of cell cycle. We use several methods to measure the cellular uptake kinetics and intracellular retention of SiNPs, including confocal laser scanning microscopy (CLSM), flow cytometry, and transmission electron microscopy (TEM). We demonstrate that SiNPs readily enter into senescent cells. Once internalized, SiNPs do not exit and accumulate in the cytoplasm for long term. Our study provides a basis for future development of NP-based tools that can detect and target senescent cells for therapy.

Degradation of Drug Delivery Nanocarriers and Payload Release: A Review of Physical Methods for Tracing Nanocarrier Biological Fate.

Nanoformulations offer multiple advantages over conventional drug delivery, enhancing solubility, biocompatibility, and bioavailability of drugs. Nanocarriers can be engineered with targeting ligands for reaching specific tissue or cells, thus reducing the side effects of payloads. Following systemic delivery, nanocarriers must deliver encapsulated drugs, usually through nanocarrier degradation. A premature degradation, or the loss of the nanocarrier coating, may prevent the drug's delivery to the targeted tissue. Despite their importance, stability and degradation of nanocarriers in biological environments are largely not studied in the literature. Here we review techniques for tracing the fate of nanocarriers, focusing on nanocarrier degradation and drug release both intracellularly and in vivo. Intracellularly, we will discuss different fluorescence techniques: confocal laser scanning microscopy, fluorescence correlation spectroscopy, lifetime imaging, flow cytometry, etc. We also consider confocal Raman microscopy as a label-free technique to trace colocalization of nanocarriers and drugs. In vivo we will consider fluorescence and nuclear imaging for tracing nanocarriers. Positron emission tomography and single-photon emission computed tomography are used for a quantitative assessment of nanocarrier and payload biodistribution. Strategies for dual radiolabelling of the nanocarriers and the payload for tracing carrier degradation, as well as the efficacy of the payload delivery in vivo, are also discussed.

Cancer Stem Cell-Inducing Media Activates Senescence Reprogramming in Fibroblasts.

Cellular senescence is a tumor-suppressive mechanism blocking cell proliferation in response to stress. However, recent evidence suggests that senescent tumor cells can re-enter the cell cycle to become cancer stem cells, leading to relapse after cancer chemotherapy treatment. Understanding how the senescence reprogramming process is a precursor to cancer stem cell formation is of great medical importance. To study the interplay between senescence, stemness, and cancer, we applied a stem cell medium (SCM) to human embryonic fibroblasts (MRC5 and WI-38) and cancer cell lines (A549 and 293T). MRC5 and WI-38 cells treated with SCM showed symptoms of oxidative stress and became senescent. Transcriptome analysis over a time course of SCM-induced senescence, revealed a developmental process overlapping with the upregulation of genes for growth arrest and the senescence-associated secretory phenotype (SASP). We demonstrate that histone demethylases jumonji domain-containing protein D3 (Jmjd3) and ubiquitously transcribed tetratricopeptide repeat, X chromosome (Utx), which operate by remodeling chromatin structure, are implicated in the senescence reprogramming process to block stem cell formation in fibroblasts. In contrast, A549 and 293T cells cultured in SCM were converted to cancer stem cells that displayed the phenotype of senescence uncoupled from growth arrest. The direct overexpression of DNA methyltransferases (Dnmt1 and Dnmt3A), ten-eleven translocation methylcytosine dioxygenases (Tet1 and Tet3), Jmjd3, and Utx proteins could activate senescence-associated beta-galactosidase (SA-ß-gal) activity in 293T cells, suggesting that epigenetic alteration and chromatin remodeling factors trigger the senescence response. Overall, our study suggests that chromatin machinery controlling senescence reprogramming is significant in cancer stem cell formation.

Total DNA Methylation Changes Reflect Random Oxidative DNA Damage in Gliomas.

DNA modifications can be used to monitor pathological processes. We have previously shown that estimating the amount of the main DNA epigenetic mark, 5-methylcytosine (m5C), is an efficient and reliable way to diagnose brain tumors, hypertension, and other diseases. Abnormal increases of reactive oxygen species (ROS) are a driving factor for mutations that lead to changes in m5C levels and cancer evolution. 8-oxo-deoxyguanosine (8-oxo-dG) is a specific marker of ROS-driven DNA-damage, and its accumulation makes m5C a hotspot for mutations. It is unknown how m5C and 8-oxo-dG correlate with the malignancy of gliomas. We analyzed the total contents of m5C and 8-oxo-dG in DNA from tumor tissue and peripheral blood samples from brain glioma patients. We found an opposite relationship in the amounts of m5C and 8-oxo-dG, which correlated with glioma grade in the way that low level of m5C and high level of 8-oxo-dG indicated increased glioma malignancy grade. Our results could be directly applied to patient monitoring and treatment protocols for gliomas, as well as bolster previous findings, suggesting that spontaneously generated ROS react with m5C. Because of the similar mechanisms of m5C and guanosine oxidation, we concluded that 8-oxo-dG could also predict glioma malignancy grade and global DNA demethylation in cancer cells.

Regulatory RNAs in Planarians.

The full scope of regulatory RNA evolution and function in epigenetic processes is still not well understood. The development of planarian flatworms to be used as a simple model organism for research has shown a great potential to address gaps in the knowledge in this field of study. The genomes of planarians encode a wide array of regulatory RNAs that function in gene regulation. Here, we review planarians as a suitable model organism for the identification and function of regulatory RNAs.

tRNA--the golden standard in molecular biology.

Transfer RNAs (tRNAs) represent a major class of RNA molecules. Their primary function is to help decode a messenger RNA (mRNA) sequence in order to synthesize protein and thus ensures the precise translation of genetic information that is imprinted in DNA. The discovery of tRNA in the late 1950's provided critical insight into a genetic machinery when little was known about the central dogma of molecular biology. In 1965, Robert Holley determined the first nucleotide sequence of alanine transfer RNA (tRNA(Ala)) which earned him the 1968 Nobel Prize in Physiology or Medicine. Today, tRNA is one of the best described and characterized biological molecules. Here we review some of the key historical events in tRNA research which led to breakthrough discoveries and new developments in molecular biology.

L-MYC Expression Maintains Self-Renewal and Prolongs Multipotency of Primary Human Neural Stem Cells.

Pre-clinical studies indicate that neural stem cells (NSCs) can limit or reverse CNS damage through direct cell replacement, promotion of regeneration, or delivery of therapeutic agents. Immortalized NSC lines are in growing demand due to the inherent limitations of adult patient-derived NSCs, including availability, expandability, potential for genetic modifications, and costs. Here, we describe the generation and characterization of a new human fetal NSC line, immortalized by transduction with L-MYC (LM-NSC008) that in vitro displays both self-renewal and multipotent differentiation into neurons, oligodendrocytes, and astrocytes. These LM-NSC008 cells were non-tumorigenic in vivo, and migrated to orthotopic glioma xenografts in immunodeficient mice. When administered intranasally, LM-NSC008 distributed specifically to sites of traumatic brain injury (TBI). These data support the therapeutic development of immortalized LM-NSC008 cells for allogeneic use in TBI and other CNS diseases.

Planarians as a model of aging to study the interaction between stem cells and senescent cells in vivo.

The depletion of stem cell pools and the accumulation of senescent cells in animal tissues are linked to aging. Planarians are invertebrate flatworms and are unusual in that their stem cells, called neoblasts, are constantly replacing old and dying cells. By eliminating neoblasts in worms via irradiation, the biological principles of aging are exposed in the absence of wound healing and regeneration, making planaria a powerful tool for aging research.

The histone demethylase jumonji coordinates cellular senescence including secretion of neural stem cell-attracting cytokines.

UNLABELLED: Jumonji domain-containing protein 3 (JMJD3/KDM6B) demethylates lysine 27 on histone H3 (H3K27me3), a repressive epigenetic mark controlling chromatin organization and cellular senescence. To better understand the functional consequences of JMJD3 its expression was investigated in brain tumor cells. Querying patient expression profile databases confirmed JMJD3 overexpression in high-grade glioma. Immunochemical staining of two glioma cell lines, U251 and U87, indicated intrinsic differences in JMJD3 expression levels that were reflected in changes in cell phenotype and variations associated with cellular senescence, including senescence-associated ß-galactosidase (SA-ß-gal) activity and the senescence-associated secretory phenotype (SASP). Overexpressing wild-type JMJD3 (JMJD3wt) activated SASP-associated genes, enhanced SA-ß-gal activity, and induced nuclear blebbing. Conversely, overexpression of a catalytically inactive dominant negative mutant JMJD3 (JMJD3mut) increased proliferation. In addition, a large number of transcripts were identified by RNA-seq as altered in JMJD3 overexpressing cells, including cancer- and inflammation-related transcripts as defined by Ingenuity Pathway Analysis. These results suggest that expression of the SASP in the context of cancer undermines normal tissue homeostasis and contributes to tumorigenesis and tumor progression. These studies are therapeutically relevant because inflammatory cytokines have been linked to homing of neural stem cells and other stem cells to tumor loci. IMPLICATIONS: This glioma study brings together actions of a normal epigenetic mechanism (JMJD3 activity) with dysfunctional activation of senescence-related processes, including secretion of SASP proinflammatory cytokines and stem cell tropism toward tumors.