The dark side of circulating nucleic acids

Free circulating or cell‐free DNA (cfDNA), possibly from dying cells that release their contents into the blood as they break down, have become of major interest as a source for noninvasive diagnostics. Recent work demonstrated the uptake of human cfDNA in mouse cells in vitro and in vivo, accompanied by the activation of a cellular DNA damage response (DDR) and the appearance of apoptotic proteins in the host cells. By acting as a source of mobile genetic elements, cfDNA could be a continuous source of DNA mutagenesis of healthy cells in the body throughout life, promoting progressive cellular aging in vivo. As such, cfDNA may causally contribute to multiple aging‐related diseases, such as cancer, diabetes, and Alzheimer's disease.     

Enhanced Expression of Fibronectin during in Vivo Cellular Aging of Human Vascular Endothelial Cells and Skin Fibroblasts

Vascular endothelial cells are thought to play an important role in human aging as their senescence and/or detachment from vascular wall contribute to arteriosclerosis and high blood pressure in elderly persons. Since fibronectin is necessary for cell attachment and spreading and its increased expression has been reported in aging fibroblasts, we checked its expression in aortic endothelial cells aged in vivo. We found that the steady-state level of fibronectin expression increases with increasing donor age, while the labeling index of cultured cells decreases with age. The increased level of fibronectin expression correlated well with an increase in cell area. To explore whether these changes were a reflection of exhaustion of proliferation potential in vivo, we examined fibronectin expression in human umbilical vein endothelial cells aging in vitro. Very similar results were obtained, supporting the idea that vascular endothelial cells age in vivo by using up division potential. When we examined the expression of fibronectin in human skin fibroblasts aged in vivo and fetal lung fibroblasts aged in vitro, we obtained similar results. In conclusion, the level of expression of fibronectin and cell size increase during in vivo and in vitro aging of both endothelial cells and fibroblasts in a coordinate manner.     

Quantification of the cellular proliferation on freshly dispersed cells from rat anterior pituitaries after in vivo and in vitro labelling with bromodeoxyuridine.

Summary The labelling index i.e., the proportion of cells in S phase of the cell cycle, has been calculated in cytospin preparations of rat anterior pituitary cells after labelling eitherin vivo orin vitro with the thymidine analogue bromodeoxyuridine (BrdU). The aims of this work were (1) to check whether enzymatic digestion interferes with the incorporation of BrdU into S phase cells and/or whether it has any deletereous effect on the immunohistochemical detection of cells that have already incorporated BrdU, and (2) to check the viability of simultaneous staining for BrdU and markers for the different types of pituitary cells in the cytospins. No statistical difference was found between the labelling index afterin vivo orin vitro labelling with BrdU. Identification of doubly-immunostained cells was straightforward and up to 40% of BrdU-labelled cells were immunopositive for pituitary hormones. It is suggested that cytospin preparations from biopsy samples may be used to study cellular proliferation without exposing the patient to the hazardous effects of BrdU infusion and without the interference of cell culture methods.     

Symposium 10: Differentiation Plasticity of Stem Cells. Direct differentiation of radial glial cells from embryonic stem cells

The major role of radial glial cells in neuronal development is to provide support and guidance for neuronal migration. In vitro, neurons, astrocytes and oligodendrocytes have also been generated from neural stem cells and embryonic stem cells, but the generation of radial glial cells in vitro has not yet been reported. Since radial glial cells can lead to neurons and astrocytes during brain development, neurogenesis and gliogenesis of stem cells in vitro may at least in part also utilize the same mechanisms. To test this hypothesis, we utilized five different clones of embryonic (ES) and embryonal carcinoma (EC) stem cell lines to investigate the differentiation of radial glial cells during in vitro neural differentiation. Here, we demonstrate that radial glial cells can be generated from ES/EC cell lines. These ES/EC cell‐derived radial glial cells are similar in morphology to radial glial cells in vivo. They also express several cytoskeletal markers that are characteristics of radial glial cells in vivo. The processes of these in vitro‐generated radial glial cells are organized into scaffolds that appear to support the migration of newly generated neurons in culture. Like radial glial cells in vivo, they appear to differentiate subsequently into astrocytes. Differentiation of radial glial cells may be a common pathway during in vitro neural differentiation of ES cells. This novel in vitro model system may facilitate the investigation of regulation of radial glial cell differentiation and its biological function. Acknowledgements: Supported by USPHS Grant NS11853 and a grant from the Children's Medical Research Foundation.     

Interest and limits of in vitro studies in renal vascular endocrinology

Various in vitro preparations have been utilized to study the cellular activity of vasoactive agents on renal cortical microvessels and on mesangial cells. The receptors and the transduction pathways of bradykinin and atrial natriuretic factor were characterized on cultured cortical vascular smooth muscle cells from the rabbit kidney. A preparation of afferent arterioles that had been freshly isolated from the rat kidney was used to study the NO-dependent regulation of renin release. The influence of endothelin and angiotensin II on mesangial cell proliferation was evaluated, using cocultures of human endothelial and mesangial cells. These appropriate in vitro preparations have provided new insights on renal vascular endocrinology. However, extrapolation of in vitro data to in vivo physiology must be cautious because the phenotype of vascular cells often changes in culture conditions.Abbreviations ANF atrial natriuretic factor - BK bradykinin - CNP C-type natriuretic peptide - ET-1 endothelin-1 - HUVEC human umbilical vein endothelial cells - IBMX isobutylmethylxanthine - NEP neutral endopeptidase - PKA protein kinase A - RCVSMC renal cortical vascular smoothmuscle cells     

Increased susceptibility to SV40 transformation with development and in vitro aging

The incidence of most cancers increases with aging. To examine whether this increased risk might be related to a higher susceptibility of older cells to neoplastic transformation, we transfected rat fibroblasts aged in vivo and in vitro with origin-defective SV40 DNA and measured the number of transformed foci. Substantial increases in the number of transformed foci were observed in cells from adult rats when compared with those of cells from embryos or weanlings. Much higher numbers of foci were also obtained at late passage, when 68% or more of the in vitro lifespan had been completed, while no foci were produced from cells at early or middle passage. To control for changes with aging in uptake, integration, or expression of exogenous DNA, parallel cultures were transfected with a G418 resistance gene. The number of G418-resistant colonies did not increase with aging and, in fact, decreased in late passage embryonic cell cultures. Therefore, increased susceptibility to SV40 transformation appears to be a feature of development and in vitro aging in rat Cells.     

Promotion of tumor development in prostate cancer by progerin

Progerin is a truncated form of lamin A. It is identified in patients with Hutchinson-Gilford progeria syndrome (HGPS), a disease characterized by accelerated aging. The contribution of progerin toward aging has been shown to be related to increased DNA damages. Since aging is one major risk factor for carcinogenesis, and genomic instability is a hallmark of malignant cancers, we investigated the expression of progerin in human cancer cells, and whether its expression contributes to carcinogenesis. Using RT-PCR and Western blotting, we detected the expression of progerin in prostate PC-3, DU145 and LNCaP cells at mRNA and protein levels. Ectopic progerin expression did not cause cellular senescence in PC-3 or MCF7 cells. PC-3 cells progerin transfectants were sensitized to DNA damage agent camptothecin (CPT); and persistent DNA damage responses were observed, which might be caused by progerin induced defective DNA damage repair. In addition, progerin transfectants were more tumorigenic in vivo than vector control cells. Our study for the first time describes the expression of progerin in a number of human cancer cell lines and its contributory role in tumorigenesis.     

TCTP Is an Androgen-Regulated Gene Implicated in Prostate Cancer

TCTP has been implicated in a plethora of important cellular processes related to cell growth, cell cycle progression, malignant transformation and inhibition of apoptosis. In addition to these intracellular functions, TCTP has extracellular functions and plays an important role in immune cells. TCTP expression was previously shown to be deregulated in prostate cancer, but its function in prostate cancer cells is largely unknown. Here we show that TCTP expression is regulated by androgens in LNCaP prostate cancer cells in vitro as well as human prostate cancer xenografts in vivo. Knockdown of TCTP reduced colony formation and increased apoptosis in LNCaP cells, implicating it as an important factor for prostate cancer cell growth. Global gene expression profiling in TCTP knockdown LNCaP cells showed that several interferon regulated genes are regulated by TCTP, suggesting that it may have a role in regulating immune function in prostate cancer. In addition, recombinant TCTP treatment increased colony formation in LNCaP cells suggesting that secreted TCTP may function as a proliferative factor in prostate cancer. These results suggest that TCTP may have a role in prostate cancer development.     

DNA polymerase α and the regulation of entry into S phase in heterokaryons

We have previously reported that the DNA polymerase α activity/unit cellular protein is decreased in latepassage (senescent) human diploid fibroblast-like (HDFL) cultures due to the cellular enlargement associated with in vitro aging. In the studies described here, we have used cell fusion technology to investigate the formal kinetic relationship between the concentration of DNA polymerase α and the rate of reinitiation of DNA synthesis in nuclei from senescent cells. Heterokaryons were derived from the fusion of senescent cells to a series of actively dividing cell types with inherently different DNA polymerase α activities per cell. A kinetic analysis revealed a first-order relationship between the entry into S phase of senescent nuclei and the concentration of DNA polymerase a activity calculated to be in heterokaryons. This result suggests that increases in cell volume may be related to the decline in proliferative activity of late-passage HDFL cells, via “dilution” of factors essential for cellular replication.     

Reduced naïve CD8+ T‐cell priming efficacy in elderly adults

Aging is associated with impaired vaccine efficacy and increased susceptibility to infectious and malignant diseases. CD8⁺ T‐cells are key players in the immune response against pathogens and tumors. In aged mice, the dwindling naïve CD8⁺ T‐cell compartment is thought to compromise the induction of de novo immune responses, but no experimental evidence is yet available in humans. Here, we used an original in vitro assay based on an accelerated dendritic cell coculture system in unfractioned peripheral blood mononuclear cells to examine CD8⁺ T‐cell priming efficacy in human volunteers. Using this approach, we report that old individuals consistently mount quantitatively and qualitatively impaired de novo CD8⁺ T‐cell responses specific for a model antigen. Reduced CD8⁺ T‐cell priming capacity in vitro was further associated with poor primary immune responsiveness in vivo. This immune deficit likely arises as a consequence of intrinsic cellular defects and a reduction in the size of the naïve CD8⁺ T‐cell pool. Collectively, these findings provide new insights into the cellular immune insufficiencies that accompany human aging.     

Enzymology in vivo using NMR and molecular genetics

Models of metabolic flux regulation are frequently based on an extrapolation of the kinetic properties of enzymes measured in vitro to the intact cell. Such an extrapolation assumes a detailed knowledge of the intracellular environment of these enzymes in terms of their free substates and effectors concentrations and possible interaction with other cellular macromolecules, which may modify their kinetic properties. These is a considerable incentive, therefore, to study the properties of enzymes directly in vivo. We have been using non-invasive NMR techniques, in conjunction with molecular genetic manipulation of enzyme levels, to study the kinetic properties of individual enzymes in vivo. We have also developed a novel strategy which has allowed us to monitor, by NMR, the ligand binding properties and mobilities of enzymes in the intact cell. This technique may also allow us to measured the diffusion coefficients of these proteins in the cell. These studies should give new insight into the properties of enzymes in vivo     

SPOCK1 promotes the invasion and metastasis of gastric cancer through Slug‐induced epithelial‐mesenchymal transition

Metastasis is a crucial impediment to the successful treatment for gastric cancer. SPOCK1 has been demonstrated to facilitate cancer metastasis in certain types of cancers; however, the role of SPOCK1 in the invasion and metastasis of gastric cancer remains elusive. SPOCK1 and epithelial‐mesenchymal transition (EMT)‐related biomarkers were detected by immunohistochemistry and Western blot in gastric cancer specimens. Other methods including stably transfected against SPOCK1 into gastric cancer cells, Western blot, migration and invasion assays in vitro and metastasis assay in vivo were also performed. The elevated expression of SPOCK1 correlates with EMT‐related markers in human gastric cancer tissue, clinical metastasis and a poor prognosis in patients with gastric cancer. In addition, knockdown of SPOCK1 expression significantly inhibits the invasion and metastasis of gastric cancer cells in vitro and in vivo, inversely, SPOCK1 overexpression results in the opposite effect. Interestingly, SPOCK1 expression has no effect on cell proliferation in vitro and in vivo. Regarding the mechanism(s) of SPOCK1‐induced cells invasion and metastasis, we prove that Slug‐induced EMT is involved in SPOCK1‐facilitating gastric cancer cells invasion and metastasis. The elevated SPOCK1 expression is closely correlated with cancer metastasis and patient survival, and SPOCK1 promotes the invasion and metastasis of gastric cancer through Slug‐mediated EMT, thereby possibly providing a novel therapeutic target for gastric cancer.     

Neutrophils induce paracrine telomere dysfunction and senescence in ROS‐dependent manner

Cellular senescence is characterized by an irreversible cell cycle arrest as well as a pro‐inflammatory phenotype, thought to contribute to aging and age‐related diseases. Neutrophils have essential roles in inflammatory responses; however, in certain contexts their abundance is associated with a number of age‐related diseases, including liver disease. The relationship between neutrophils and cellular senescence is not well understood. Here, we show that telomeres in non‐immune cells are highly susceptible to oxidative damage caused by neighboring neutrophils. Neutrophils cause telomere dysfunction both in vitro and ex vivo in a ROS‐dependent manner. In a mouse model of acute liver injury, depletion of neutrophils reduces telomere dysfunction and senescence. Finally, we show that senescent cells mediate the recruitment of neutrophils to the aged liver and propose that this may be a mechanism by which senescence spreads to surrounding cells. Our results suggest that interventions that counteract neutrophil‐induced senescence may be beneficial during aging and age‐related disease.     

Isolation and therapeutic potential of human haemopoietic stem cells

The haemopoietic stem cell (HSC) has long been regarded as an archetypal, tissue specific, stem cell, capable of completely regenerating haemopoiesis after myeloablation. It has proved relatively easy to harvest HSC, from bone marrow or peripheral blood. In turn, isolation of these cells has allowed therapeutic stem cell transplantation protocols to be developed, that capitalise on their prodigious self renewal and proliferative capabilities. Ex vivo approaches have been described to isolate, genetically manipulateand expand pluripotent stem cell subsets. These techniques have been crucial to the development of gene therapy, and may allow adults to enjoy the potential advantages of cord blood transplantation. Recently, huge conceptual changes have occurred in stem cell biology. In particular, the dogma that, in adults, stem cells are exclusively tissue restricted has been questioned and there is great excitement surrounding the potential plasticity of these cells, with the profound implications that this has, for developing novel cellular therapies. Mesenchymal stem cells, multipotent adult progenitor cells and embryonic stem cells are potential sources of cells for transplantation purposes. These cells may be directed toproduce HSC, in vitro and in the future may be used for therapeutic, or drug development, purposes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Pluripotency of male germline stem cells

The ethical issues and public concerns regarding the use of embryonic stem (ES) cells in human therapy have motivated considerable research into the generation of pluripotent stem cell lines from non-embryonic sources. Numerous reports have shown that pluripotent cells can be generated and derived from germline stem cells (GSCs) in mouse and human testes during in vitro cultivation. The gene expression patterns of these cells are similar to those of ES cells and show the typical self-renewal and differentiation patterns of pluripotent cells in vivo and in vitro. However, the mechanisms underlying the spontaneous dedifferentiation of GSCs remain to be elucidated. Studies to identify master regulators in this reprogramming process are of critical importance for understanding the gene regulatory networks that sustain the cellular status of these cells. The results of such studies would provide a theoretical background for the practical use of these cells in regenerative medicine. Such studies would also help elucidate the molecular mechanisms underlying certain diseases, such as testicular germ cell tumors.     

Downregulation of developmentally regulated endothelial cell locus-1 inhibits the growth of colon cancer

Developmentally regulated endothelial cell locus-1 (Del1) is an embryonic angiogenic factor expressed in early embryonic endothelial cells, but recently has been found to be expressed in some forms of cancers including colon and breast cancers, and melanoma, and human cancer cell lines. Overexpression of Del1 accelerates tumor growth by enhancing vascular formation, implying Del1 may be a potential target for anti-angiogenic cancer therapy. The study aims to investigate whether downregulation of Del1 could inhibit the growth of tumors established in nude Balb/c mice by subcutaneous implantation of human LS-174T colon cancer cells. The shRNA expression vectors targeting human Del1, and vascular endothelial growth factor (VEGF) were constructed. Gene transfection of Del1-shRNA downregulated expression of Del1 in LS-174T cells in vivo and in vitro, but did not alter the proliferative or survival properties of cells in vitro. Gene transfection of VEGF-shRNA downregulated expression of both VEGF and Del1 in LS-174T cells in vivo and in vitro. Both Del1-shRNA and VEGF-shRNA gene therapies exhibited anti-tumor activities and they also showed a synergistic effect in suppressing growth of colon tumors by anti-angiogenesis and anti-proliferation. Although further investigation to clarify the mechanisms explaining the role of Del1 in tumor growth, and the interaction between VEGF and Del1, is required, the results indicate that downregulation of Del1 presents a potent therapeutic strategy to combat colon cancer.     

Perinatal sources of mesenchymal stem cells: Wharton’s jelly,amnion and chorion

Recently, stem cell biology has become an interesting topic, especially in the context of treating diseases and injuries using transplantation therapy. Several varieties of human stem cells have been isolated and identified in vivo and in vitro. Ideally, stem cells for regenerative medical application should be found in abundant quantities, harvestable in a minimally invasive procedure, then safely and effectively transplanted to either an autologous or allogenic host. The two main groups of stem cells, embryonic stem cells and adult stem cells, have been expanded to include perinatal stem cells. Mesenchymal stem cells from perinatal tissue may be particularly useful in the clinic for autologous transplantation for fetuses and newborns, and after banking in later stages of life, as well as for in utero transplantation in case of genetic disorders.     

Exploring oxidative modifications of tyrosine: An update on mechanisms of formation,advances in analysis and biological consequences

Abstract

Protein oxidation is increasingly recognised as an important modulator of biochemical pathways controlling both physiological and pathological processes. While much attention has focused on cysteine modifications in reversible redox signalling, there is increasing evidence that other protein residues are oxidised in vivo with impact on cellular homeostasis and redox signalling pathways. A notable example is tyrosine, which can undergo a number of oxidative post-translational modifications to form 3-hydroxy-tyrosine, tyrosine crosslinks, 3-nitrotyrosine and halogenated tyrosine, with different effects on cellular functions. Tyrosine oxidation has been studied extensively in vitro, and this has generated detailed information about the molecular mechanisms that may occur in vivo. An important aspect of studying tyrosine oxidation both in vitro and in biological systems is the ability to monitor the formation of oxidised derivatives, which depends on a variety of analytical techniques. While antibody-dependent techniques such as ELISAs are commonly used, these have limitations, and more specific assays based on spectroscopic or spectrometric techniques are required to provide information on the exact residues modified and the nature of the modification. These approaches have helped understanding of the consequences of tyrosine oxidation in biological systems, especially its effects on cell signalling and cell dysfunction, linking to roles in disease. There is mounting evidence that tyrosine oxidation processes are important in vivo and can contribute to cellular pathology.     

Retinal pigment epithelial cell multinucleation in the aging eye – a mechanism to repair damage and maintain homoeostasis

Retinal pigment epithelial (RPE) cells are central to retinal health and homoeostasis. Dysfunction or death of RPE cells underlies many age‐related retinal degenerative disorders particularly age‐related macular degeneration. During aging RPE cells decline in number, suggesting an age‐dependent cell loss. RPE cells are considered to be postmitotic, and how they repair damage during aging remains poorly defined. We show that RPE cells increase in size and become multinucleate during aging in C57BL/6J mice. Multinucleation appeared not to be due to cell fusion, but to incomplete cell division, that is failure of cytokinesis. Interestingly, the phagocytic activity of multinucleate RPE cells was not different from that of mononuclear RPE cells. Furthermore, exposure of RPE cells in vitro to photoreceptor outer segment (POS), particularly oxidized POS, dose‐dependently promoted multinucleation and suppressed cell proliferation. Both failure of cytokinesis and suppression of proliferation required contact with POS. Exposure to POS also induced reactive oxygen species and DNA oxidation in RPE cells. We propose that RPE cells have the potential to proliferate in vivo and to repair defects in the monolayer. We further propose that the conventionally accepted ‘postmitotic’ status of RPE cells is due to a modified form of contact inhibition mediated by POS and that RPE cells are released from this state when contact with POS is lost. This is seen in long‐standing rhegmatogenous retinal detachment as overtly proliferating RPE cells (proliferative vitreoretinopathy) and more subtly as multinucleation during normal aging. Age‐related oxidative stress may promote failure of cytokinesis and multinucleation in RPE cells.