Heterogeneity of adult masseter muscle satellite cells with cardiomyocyte differentiation potential.

Although resident cardiac stem cells have been reported, regeneration of functional cardiomyocytes (CMs) remains a challenge. The present study identifies an alternative progenitor source for CM regeneration without the need for genetic manipulation or invasive heart biopsy procedures. Unlike limb skeletal muscles, masseter muscles (MM) in the mouse head are developed from Nkx2-5 mesodermal progenitors. Adult masseter muscle satellite cells (MMSCs) display heterogeneity in developmental origin and cell phenotypes. The heterogeneous MMSCs that can be characterized by cell sorting based on stem cell antigen-1 (Sca1) show different lineage potential. While cardiogenic potential is preserved in Sca1+ MMSCs as shown by expression of cardiac progenitor genes (including Nkx2-5), skeletal myogenic capacity is maintained in Sca1- MMSCs with Pax7 expression. Sca1+ MMSC-derived beating cells express cardiac genes and exhibit CM-like morphology. Electrophysiological properties of MMSC-derived CMs are demonstrated by calcium transients and action potentials. These findings show that MMSCs could serve as a novel cell source for cardiomyocyte replacement.

Next-generation sequencing technologies: breaking the sound barrier of human genetics.

Demand for new technologies that deliver fast, inexpensive and accurate genome information has never been greater. This challenge has catalysed the rapid development of advances in next-generation sequencing (NGS). The generation of large volumes of sequence data and the speed of data acquisition are the primary advantages over previous, more standard methods. In 2013, the Food and Drug Administration granted marketing authorisation for the first high-throughput NG sequencer, Illumina's MiSeqDx, which allowed the development and use of a large number of new genome-based tests. Here, we present a review of template preparation, nucleic acid sequencing and imaging, genome assembly and alignment approaches as well as recent advances in current and near-term commercially available NGS instruments. We also outline the broad range of applications for NGS technologies and provide guidelines for platform selection to best address biological questions of interest. DNA sequencing has revolutionised biological and medical research, and is poised to have a similar impact on the practice of medicine. This tool is but one of an increasing arsenal of developing tools that enhance our capabilities to identify, quantify and functionally characterise the components of biological networks that keep us healthy or make us sick. Despite advances in other 'omic' technologies, DNA sequencing and analysis, in many respects, have played the leading role to date. The new technologies provide a bridge between genotype and phenotype, both in man and model organisms, and have revolutionised how risk of developing a complex human disease may be assessed. The generation of large DNA sequence data sets is producing a wealth of medically relevant information on a large number of individuals and populations that will potentially form the basis of truly individualised medical care in the future.

Whole genome sequencing of glioblastoma multiforme identifies multiple structural variations involved in EGFR activation.

Next generation sequencing has become a powerful tool in dissecting and identifying mutations and genomic structural variants that accompany tumourigenesis. Sequence analysis of glioblastoma multiforme (GBM) illustrates the ability to rapidly identify mutations that may affect phenotype. Approximately 50% of human GBMs overexpress epidermal growth factor receptor (EGFR) which renders the EGFR protein a compelling therapeutic target. In brain tumours, attempts to target EGFR as a cancer therapeutic, however, have achieved little or no benefit. The mechanisms that drive therapeutic resistance to EGFR inhibitors in brain tumours are not well defined, and drug resistance contributes to the deadly and aggressive nature of the disease. Whole genome sequencing of four primary GBMs revealed multiple pathways by which EGFR protein abundance becomes deregulated in these tumours and will guide the development of new strategies for treating EGFR overexpressing tumours. Each of the four tumours displayed a different mechanism leading to increased EGFR protein levels. One mechanism is mediated by gene amplification and tandem duplication of the kinase domain. A second involves an intragenic deletion that generates a constitutively active form of the protein. A third combines the loss of a gene which encodes a protein that regulates EGFR abundance as well as an miRNA that modulates EGFR expression. A fourth mechanism entails loss of an ubiquitin ligase docking site in the C-terminal part of the protein whose absence inhibits turnover of the receptor.

Nardilysin-regulated scission mechanism activates polo-like kinase 3 to suppress the development of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) develops through step-wise genetic and molecular alterations including Kras mutation and inactivation of various apoptotic pathways. Here, we find that development of apoptotic resistance and metastasis of KrasG12D-driven PDAC in mice is accelerated by deleting Plk3, explaining the often-reduced Plk3 expression in human PDAC. Importantly, a 41-kDa Plk3 (p41Plk3) that contains the entire kinase domain at the N-terminus (1-353 aa) is activated by scission of the precursor p72Plk3 at Arg354 by metalloendopeptidase nardilysin (NRDC), and the resulting p32Plk3 C-terminal Polo-box domain (PBD) is removed by proteasome degradation, preventing the inhibition of p41Plk3 by PBD. We find that p41Plk3 is the activated form of Plk3 that regulates a feed-forward mechanism to promote apoptosis and suppress PDAC and metastasis. p41Plk3 phosphorylates c-Fos on Thr164, which in turn induces expression of Plk3 and pro-apoptotic genes. These findings uncover an NRDC-regulated post-translational mechanism that activates Plk3, establishing a prototypic regulation by scission mechanism.

The human DEK oncogene regulates DNA damage response signaling and repair.

The human DEK gene is frequently overexpressed and sometimes amplified in human cancer. Consistent with oncogenic functions, Dek knockout mice are partially resistant to chemically induced papilloma formation. Additionally, DEK knockdown in vitro sensitizes cancer cells to DNA damaging agents and induces cell death via p53-dependent and -independent mechanisms. Here we report that DEK is important for DNA double-strand break repair. DEK depletion in human cancer cell lines and xenografts was sufficient to induce a DNA damage response as assessed by detection of γH2AX and FANCD2. Phosphorylation of H2AX was accompanied by contrasting activation and suppression, respectively, of the ATM and DNA-PK pathways. Similar DNA damage responses were observed in primary Dek knockout mouse embryonic fibroblasts (MEFs), along with increased levels of DNA damage and exaggerated induction of senescence in response to genotoxic stress. Importantly, Dek knockout MEFs exhibited distinct defects in non-homologous end joining (NHEJ) when compared to their wild-type counterparts. Taken together, the data demonstrate new molecular links between DEK and DNA damage response signaling pathways, and suggest that DEK contributes to DNA repair.

The novel mouse Polo-like kinase 5 responds to DNA damage and localizes in the nucleolus.

Polo-like kinases (Plk1-4) are emerging as an important class of proteins involved in many aspects of cell cycle regulation and response to DNA damage. Here, we report the cloning of a fifth member of the polo-like kinase family named Plk5. DNA and protein sequence analyses show that Plk5 shares more similarities with Plk2 and Plk3 than with Plk1 and Plk4. Consistent with this observation, we show that mouse Plk5 is a DNA damage inducible gene. Mouse Plk5 protein localizes predominantly to the nucleolus, and deletion of a putative nucleolus localization signal (NoLS) within its N-terminal moiety disrupts its nucleolar localization. Ectopic expression of Plk5 leads to cell cycle arrest in G1, decreased DNA synthesis, and to apoptosis, a characteristic it shares with Plk3. Interestingly, in contrast to mouse Plk5 gene, the sequence of human Plk5 contains a stop codon that produces a truncated protein lacking part of the kinase domain.

Mice with the CHEK2*1100delC SNP are predisposed to cancer with a strong gender bias.

The CHEK2 kinase (Chk2 in mouse) is a member of a DNA damage response pathway that regulates cell cycle arrest at cell cycle checkpoints and facilitates the repair of dsDNA breaks by a recombination-mediated mechanism. There are numerous variants of the CHEK2 gene, at least one of which, CHEK2*1100delC (SNP), associates with breast cancer. A mouse model in which the wild-type Chk2 has been replaced by a Chk2*1100delC allele was tested for elevated risk of spontaneous cancer and increased sensitivity to challenge by a carcinogenic compound. Mice homozygous for Chk2*1100delC produced more tumors than wild-type mice, whereas heterozygous mice were not statistically different. When fractionated by gender, however, homozygous and heterozygous mice developed spontaneous tumors more rapidly and to a far greater extent than wild-type mice, indicative of a marked gender bias in mice harboring the variant allele. Consistent with our previous data showing elevated genomic instability in mouse embryonic fibroblasts (MEFs) derived from mice homozygous for Chk2*1100delC, the level of Cdc25A was elevated in heterozygous and homozygous MEFs and tumors. When challenged with the carcinogen 7,12-dimethylbenz[a]anthracene, all mice, regardless of genotype, had a reduced lifespan. Latency for mammary tumorigenesis was reduced significantly in mice homozygous for Chk2*1100delC but unexpectedly increased for the development of lymphomas. An implication from this study is that individuals who harbor the variant CHEK2*1100delC allele not only are at an elevated risk for the development of cancer but also that this risk can be further increased as a result of environmental exposure.

Absence of polo-like kinase 3 in mice stabilizes Cdc25A after DNA damage but is not sufficient to produce tumors.

The polo-like kinases (Plks1-5) are emerging as an important class of proteins involved in many facets of cell cycle regulation and response to DNA damage and stress. Here we show that Plk3 phosphorylates the key cell cycle protein phosphatase Cdc25A on two serine residues in its cyclinB/cdk1 docking domain and regulates its stability in response to DNA damage. We generated a Plk3 knock-out mouse and show that Cdc25A protein from Plk3-deficient cells is less susceptible to DNA damage-mediated degradation than cells with functional Plk3. We also show that absence of Plk3 correlates with loss of the G1/S cell cycle checkpoint. However, neither this compromised DNA damage checkpoint nor reduced susceptibility to proteasome-mediated degradation after DNA damage translated into a significant increase in tumor incidence in the Plk3-deficient mice.

Ionizing radiation is a potent inducer of mitotic recombination in mouse embryonic stem cells.

Maintenance of genomic integrity in embryonic cells is pivotal to proper embryogenesis, organogenesis and to the continuity of species. Cultured mouse embryonic stem cells (mESCs), a model for early embryonic cells, differ from cultured somatic cells in their capacity to remodel chromatin, in their repertoire of DNA repair enzymes, and in the regulation of cell cycle checkpoints. Using 129XC3HF1 mESCs heterozygous for Aprt, we characterized loss of Aprt heterozygosity after exposure to ionizing radiation. We report here that the frequency of loss of heterozygosity mutants in mESCs can be induced several hundred-fold by exposure to 5-10Gy of X-rays. This induction is 50-100-fold higher than the induction reported for mouse adult or embryonic fibroblasts. The primary mechanism underlying the elevated loss of heterozygosity after irradiation is mitotic recombination, with lesser contributions from deletions and gene conversions that span Aprt. Aprt point mutations and epigenetic inactivation are very rare in mESCs compared to fibroblasts. Mouse ESCs, therefore, are distinctive in their response to ionizing radiation and studies of differentiated cells may underestimate the mutagenic effects of ionizing radiation on ESC or other stem cells. Our findings are important to understanding the biological effects of ionizing radiation on early development and carcinogenesis.

Treatment of osteoporosis with a modified zeolite shows beneficial effects in an osteoporotic rat model and a human clinical trial.

The severity of osteoporosis in humans manifests in its high incidence and by its complications that diminish quality of life. A societal consequence of osteoporosis is the substantial burden that it inflicts upon patients and their families. Several bone-modifying drugs have been prescribed to patients with osteoporosis. However, evidence for their anti-fracture efficacy remains inconclusive. To the contrary, long-term use of anti-osteoporotic drugs such as bisphosphonates and Denosumab, an RANKL inhibitor, have resulted in adverse events. We now present an alternative and adjuvant approach for treatment of osteoporosis. The data derive from in vivo studies in an ovariectomized rat model and from a randomized double blind, placebo-controlled human clinical study. Both studies involved treatment with Panaceo Micro Activation (PMA)-zeolite-clinoptilolite, a defined cation exchange clinoptilolite, which clearly improved all bone histomorphometric parameters examined from ovariectomized animals, indicative for increased bone formation. Moreover, intervention with PMA-zeolite-clinoptilolite for one year proved safe in humans. Furthermore, patients treated with PMA-zeolite-clinoptilolite showed an increase in bone mineral density, an elevated level of markers indicative of bone formation, a significant reduction in pain, and significantly improved quality of life compared with patients in the control (placebo) group. These encouraging positive effects of PMA-zeolite-clinoptilolite on bone integrity and on osteoporosis warrant further evaluation of treatment with PMA-zeolite-clinoptilolite as a new alternative adjuvant therapy for osteoporosis.

Preservation of genomic integrity in mouse embryonic stem cells.

Embryonic stem (ES) cells and germ cells have the potential to give rise to an entire organism. A common requirement is that both must have very robust mechanisms to preserve the integrity of their genomes. This is particularly true since somatic cells have very high mutation frequencies approaching 10-4 in vivo that would lead to unacceptable levels of fetal lethality and congenital defects. Notably, between 70% and 80% of mutational events monitored at a heterozygous endogenous selectable marker were loss of heterozygosity due to mitotic recombination, a mechanism that affects multiple heterozygous loci between the reporter gene and the site of crossing over. This chapter examines three mechanisms by which mouse embryonic stem cells preserve their genomic integrity. The first entails suppression of mutation and recombination between chromosome homologues by two orders of magnitude when compared with isogenic mouse embryo fibroblasts which had a mutation frequency similar to that seen in adult somatic cells. The second renders mouse ES cells hypersensitive to environmental challenge and eliminates damaged cells from the self-renewing population. Mouse ES cells lack a G1 checkpoint so that cells damaged by exogenous insult such as ionizing radiation do not arrest at the G1/S phase checkpoint but progress into the S phase where the damaged DNA is replicated, the damage exacerbated and the cells driven to apoptosis. The third mechanism examines how mouse ES cells repair double strand DNA breaks. Somatic cells predominantly utilize error prone nonhomologous end joining which, from a teleological perspective, would be disadvantageous for ES cells since it would promote accumulation of mutations. When ES cells were tested for the preferred pathway of double strand DNA break repair, they predominantly utilized the high fidelity homology-mediated repair pathway, thereby minimizing the incurrence of mutations during the repair process. When mouse ES cells are induced to differentiate, the predominant repair pathway switches from homology-mediated repair to nonhomologous end joining that is characteristic of somatic cells.

BRUCE preserves genomic stability in the male germline of mice.

BRUCE is a DNA damage response protein that promotes the activation of ATM and ATR for homologous recombination (HR) repair in somatic cells, making BRUCE a key protector of genomic stability. Preservation of genomic stability in the germline is essential for the maintenance of species. Here, we show that BRUCE is required for the preservation of genomic stability in the male germline of mice, specifically in spermatogonia and spermatocytes. Conditional knockout of Bruce in the male germline leads to profound defects in spermatogenesis, including impaired maintenance of spermatogonia and increased chromosomal anomalies during meiosis. Bruce-deficient pachytene spermatocytes frequently displayed persistent DNA breaks. Homologous synapsis was impaired, and nonhomologous associations and rearrangements were apparent in up to 10% of Bruce-deficient spermatocytes. Genomic instability was apparent in the form of chromosomal fragmentation, translocations, and synapsed quadrivalents and hexavalents. In addition, unsynapsed regions of rearranged autosomes were devoid of ATM and ATR signaling, suggesting an impairment in the ATM- and ATR-dependent DNA damage response of meiotic HR. Taken together, our study unveils crucial functions for BRUCE in the maintenance of spermatogonia and in the regulation of meiotic HR-functions that preserve the genomic stability of the male germline.

DNA repair in murine embryonic stem cells and differentiated cells.

Embryonic stem (ES) cells are rapidly proliferating, self-renewing cells that have the capacity to differentiate into all three germ layers to form the embryo proper. Since these cells are critical for embryo formation, they must have robust prophylactic mechanisms to ensure that their genomic integrity is preserved. Indeed, several studies have suggested that ES cells are hypersensitive to DNA damaging agents and readily undergo apoptosis to eliminate damaged cells from the population. Other evidence suggests that DNA damage can cause premature differentiation in these cells. Several laboratories have also begun to investigate the role of DNA repair in the maintenance of ES cell genomic integrity. It does appear that ES cells differ in their capacity to repair damaged DNA compared to differentiated cells. This minireview focuses on repair mechanisms ES cells may use to help preserve genomic integrity and compares available data regarding these mechanisms with those utilized by differentiated cells.

X-rays induce distinct patterns of somatic mutation in fetal versus adult hematopoietic cells.

There are a variety of mechanisms and pathways whereby cells safeguard their genomes in the face of environmental insults that damage DNA. Whether each of these pathways is equally robust at specific developmental stages in mammals and whether they are also modulated in a tissue-specific manner, however, are unclear. Here, we report that ionizing radiation (IR) produces different types of somatic mutations in fetal cells compared with adult cells of the same lineage. While 1 Gy of X-ray significantly induced intragenic point mutations in T cells of adult mice, no point mutational effect was observed when applied to fetuses. Fetal exposure to IR, on the other hand, led to a significant elevation of mitotic recombination in T cells, which was not observed in adults. Base excision repair (BER) activity was significantly lower in fetal hematopoietic cells than in adult cells, due to a low level of DNA polymerase beta, the rate-limiting enzyme in BER. In fetal hematopoietic cells, this low BER activity, together with a high rate of proliferation, causes X-ray-induced DNA lesions, such as base damage, single strand breaks and double strand breaks, to be repaired by homologous recombination, which we observe as mitotic recombination. Higher BER activity and a relatively lower rate of cell proliferation likely contribute to the significant induction of DNA point mutations in adults. Thus, the mutational response to IR is at least partly determined by the availability of specific repair pathways and other developmentally regulated phenotypes, such as mitotic index.

Loss of microRNA-128 promotes cardiomyocyte proliferation and heart regeneration.

The goal of replenishing the cardiomyocyte (CM) population using regenerative therapies following myocardial infarction (MI) is hampered by the limited regeneration capacity of adult CMs, partially due to their withdrawal from the cell cycle. Here, we show that microRNA-128 (miR-128) is upregulated in CMs during the postnatal switch from proliferation to terminal differentiation. In neonatal mice, cardiac-specific overexpression of miR-128 impairs CM proliferation and cardiac function, while miR-128 deletion extends proliferation of postnatal CMs by enhancing expression of the chromatin modifier SUZ12, which suppresses p27 (cyclin-dependent kinase inhibitor) expression and activates the positive cell cycle regulators Cyclin E and CDK2. Furthermore, deletion of miR-128 promotes cell cycle re-entry of adult CMs, thereby reducing the levels of fibrosis, and attenuating cardiac dysfunction in response to MI. These results suggest that miR-128 serves as a critical regulator of endogenous CM proliferation, and might be a novel therapeutic target for heart repair.

An ageing question: do embryonic stem cells protect their genomes?

Many physiological and cellular processes contribute to the ageing of individuals. One hypothesis argues that the genomes of somatic cells accumulate mutations, which, in turn, alter the metabolism of the cells and contribute to the ageing process. The frequency of somatic mutation approaches 10(-4) and the majority of mutagenic events at heterozygous loci is due to loss of heterozygosity as a consequence of mitotic recombination. A corollary to the argument that somatic cells accumulate mutations is that cells of the germ line and ES cells have a greater requirement for maintaining the integrity of their genomes. In the former case, a high somatic mutation frequency predicts an increase in somatic disease, which limits our lifespan. The corollary is that cells of the germline and ES cells must minimize the mutational burden to limit the frequency of congenital disease and to ensure the proper transmission of undamaged DNA to the gene pool. This report describes two mechanisms utilized by murine ES cells to minimize DNA damage within the proliferative pool. In the first case, murine ES cells display a frequency of mutation and mitotic recombination that is about 100-fold lower than that observed in somatic cells. Second, ES cells lack a G1 checkpoint following DNA damage. When subjected to ionizing radiation, the fraction of apoptotic cells increases to about 40%. Ectopic expression of Chk2 is sufficient to establish a G1 arrest and the concomitant protection from cell death.

The breast cancer susceptibility allele CHEK2*1100delC promotes genomic instability in a knock-in mouse model.

Allelic variants of CHEK2 contribute to an elevated risk for human breast cancer and possibly other cancer types. In particular, the CHEK2*1100delC polymorphic variant has been identified as a low-penetrance breast cancer susceptibility allele in breast cancer families with wild type BRCA1 and BRCA2. To better understand the molecular basis by which this allele increases risk for disease, we have generated a mouse in which the wild type CHEK2 (Chk2 in mouse) allele has been replaced with the 1100delC variant. Mouse embryo fibroblasts (MEFs) derived from these mice have an altered cell cycle profile in which a far greater proportion of cells are in S-phase and in G2 (4N) compared with wild type cells. The mutant cells show signs of spontaneous genomic instability as indicated by polyploidy and an increase in DNA double strand breaks.

Cancer Immunotherapy: Whence and Whither.

The current concepts and practice of cancer immunotherapy evolved from classical experiments that distinguished "self" from "non-self" and the finding that humoral immunity is complemented by cellular immunity. Elucidation of the biology underlying immune checkpoints and interactions between ligands and ligand receptors that govern the immune system's ability to recognize tumor cells as foreign has led to the emergence of new strategies that mobilize the immune system to reverse this apparent tolerance. Some of these approaches have led to new therapies such as the use of mAbs to interfere with the immune checkpoint. Others have exploited molecular technologies to reengineer a subset of T cells to directly engage and kill tumor cells, particularly those of B-cell malignancies. However, before immunotherapy can become a more effective method of cancer care, there are many challenges that remain to be addressed and hurdles to overcome. Included are manipulation of tumor microenvironment (TME) to enhance T effector cell infiltration and access to the tumor, augmentation of tumor MHC expression for adequate presentation of tumor associated antigens, regulation of cytokines and their potential adverse effects, and reduced risk of secondary malignancies as a consequence of mutations generated by the various forms of genetic engineering of immune cells. Despite these challenges, the future of immunotherapy as a standard anticancer therapy is encouraging. Mol Cancer Res; 15(6); 635-50. ©2017 AACR.

Towards precision prevention: Technologies for identifying healthy individuals with high risk of disease.

The rise of advanced technologies for characterizing human populations at the molecular level, from sequence to function, is shifting disease prevention paradigms toward personalized strategies. Because minimization of adverse outcomes is a key driver for treatment decisions for diseased populations, developing personalized therapy strategies represent an important dimension of both precision medicine and personalized prevention. In this commentary, we highlight recently developed enabling technologies in the field of DNA damage, DNA repair, and mutagenesis. We propose that omics approaches and functional assays can be integrated into population studies that fuse basic, translational and clinical research with commercial expertise in order to accelerate personalized prevention and treatment of cancer and other diseases linked to aberrant responses to DNA damage. This collaborative approach is generally applicable to efforts to develop data-driven, individualized prevention and treatment strategies for other diseases. We also recommend strategies for maximizing the use of biological samples for epidemiological studies, and for applying emerging technologies to clinical applications.

The Plk3-Cdc25 circuit.

Polo-like kinases (Plks) are key regulators of the cell cycle, especially in the G2 phase and mitosis. They are incorporated into signaling networks that regulate many aspects of the cell cycle, including but not limited to centrosome maturation and separation, mitotic entry, chromosome segregation, mitotic exit, and cytokinesis. The Plks have well conserved 30-amino-acid elements, designated polo boxes (PBs), located in their carboxyl-termini, which with their flanking regions constitute a functional Polo-box domain (PBD). Members of the Plk family exist in a variety of organisms including Polo in Drosophila melanogaster; Cdc5 in Saccharomyces cerevisiae; Plo1 in Schizosaccharomyces pombe; Plx1 in Xenopus laevis; and Plk1, Snk/Plk2, Fnk/Prk/Plk3, and Sak in mammals. Polo, Cdc5, and Plo1 are essential for viability. The Plks can be separated into two groups according to their functions. The first group (Polo, Cdc5, plo1, Plx1, and Plk1) primarily performs mitotic functions, whereas the second group (Plk2 and Plk3) appears to have additional functions during the G1, S, and G2 phases of the cell cycle. Several contributions to this issue will discuss different aspects of Plk involvement in cell-cycle regulation. This review, therefore, will focus on the role of Plk3 in regulating Cdc25 phosphatase function and its effect on the cell cycle.