Regulation of Telomere Length and Organisation in Human Skin Cells in vitro and in vivo

Telomeres are specialized DNA-protein structures at the ends of the linear chromosomes that form protective caps. They are composed of multi-fold double-stranded 5’-TTAGGG-3’ repeats and a 3’ single stranded overhang that loops back and invades the duplex region. The so called T-loop structure is stabilized by a number of associated proteins that protect the DNA against degradation and hinders the cellular machinery to recognize the ends as broken DNA, thus being essential for chromosomal integrity. Investigating the three-dimensional (3D) telomere distribution we now show that telomeres of the immortal HaCaT keratinocytes are distributed in distinct non-overlapping territories within the inner third of the nuclear space in interphase cells and extend more widely during mitosis. This distinct localization is abrogated in a HaCaT variant that constitutively expresses the c-Myc onco-protein. Telomeres in HaCaT-myc cells form aggregates (TAs) that are accompanied by an overall irregular telomere distribution in interphase. Since this TA formation also leads to clustering of the respective chromosomes and TA formation is present during mitosis, TAs most likely contribute to genomic instability by forcing abnormal chromosome segregation. As a first step to approach the mechanism of TA formation we compared the difference in nuclear protein expression between HaCaT and HaCaT-myc cells by two-dimensional polyacrylamide gel electrophoresis. Out of 30 differentially expressed proteins, the most promising candidate was Matrin 3, a nuclear matrix protein that, being reduced in HaCaT-myc cells, suggests for a mechanism involving incorrect adhesion to the nuclear matrix. To investigate the role of telomere loss for skin aging in situ, we developed a 3D deconvolution microscopy based Q-FISH/immunofluorescence technique on individual cells in tissue sections. When investigating skin from different-age donors, we found that similar as for dermal fibroblasts and another non-proliferative cell type in the epidermis, the melanocytes, also the epidermal keratinocytes only show a minimal age-dependent telomere decline. Thus age-dependent telomere loss appears largely neglectable. However, we found significant inter-personal differences and most strikingly, intra-personal variations in telomere lengths between similar sites of the epidermis. Moreover, in 10 of 30 samples of normal skin, preferentially from sun exposed sites of elderly donors, we identified regions within otherwise normal looking epidermis with significantly shorter telomeres. Though size and number of these micro-lesions as well as amount of telomere shortening varied, all enclosed various basal and suprabasal differentiated cells. Most importantly, they were all characterized by expression of the p53 tumour suppressor gene and 53BP1 foci co-localizing with telomeres. Since the latter are representative for DNA double strand breaks and when co-localized with telomeres represent critically short uncapped telomeres, these date demonstrate that in these micro-lesions the telomeres are dysfunctional and likely represent stages of genomic instability. Such distinct areas can only be maintained when damage has occurred in a stem cell. We, therefore, postulate that damage did not cause cell death but was repaired and lead to a decreased telomere length. This reduced telomere length was then transmitted to the daughter cells. Thus each micro-lesions most likely represents the space of one stem cell compartment. We also identified shorter telomeres in skin from heavily sun-exposed individuals and in several skin sections in sites closer to the surface (outside) as compared to more protected areas of the epidermis (deeper parts of the rete ridges, deeper parts of the hair follicles). Since we further show that in skin from 16 volunteers irradiated with UVA, UVB, or a combination of UVA and UVB, distinct telomere shortening was visible already 3 days post irradiation, UV radiation is clearly a responsible factor for accelerated telomere loss in human skin. Another factor may be forced oxidative damage because also chronic and to a lesser extend acute wounds showed distinct telomere shortening. Finally, we demonstrate for the first time a population of rare cells within the epidermis which have the “longest” telomeres and which likely represent the stem cells. These cells are distributed rather randomly throughout the basal layer. Furthermore, when cultured and replated in organotypic cultures they re-establish as stem cells in the newly developing epidermis. Thus, telomere length is a valuable marker for stem cells and it is damage rather than replication-dependent telomere shortening that leads to significant shortening and potential sites of genomic instability in human skin

Aging and Replicative Senescence Have Related Effects on Human Stem and Progenitor Cells

The regenerative potential diminishes with age and this has been ascribed to functional impairments of adult stem cells. Cells in culture undergo senescence after a certain number of cell divisions whereby the cells enlarge and finally stop proliferation. This observation of replicative senescence has been extrapolated to somatic stem cells in vivo and might reflect the aging process of the whole organism. In this study we have analyzed the effect of aging on gene expression profiles of human mesenchymal stromal cells (MSC) and human hematopoietic progenitor cells (HPC). MSC were isolated from bone marrow of donors between 21 and 92 years old. 67 genes were age-induced and 60 were age-repressed. HPC were isolated from cord blood or from mobilized peripheral blood of donors between 27 and 73 years and 432 genes were age-induced and 495 were age-repressed. The overlap of age-associated differential gene expression in HPC and MSC was moderate. However, it was striking that several age-related gene expression changes in both MSC and HPC were also differentially expressed upon replicative senescence of MSC in vitro. Especially genes involved in genomic integrity and regulation of transcription were age-repressed. Although telomerase activity and telomere length varied in HPC particularly from older donors, an age-dependent decline was not significant arguing against telomere exhaustion as being causal for the aging phenotype. These studies have demonstrated that aging causes gene expression changes in human MSC and HPC that vary between the two different cell types. Changes upon aging of MSC and HPC are related to those of replicative senescence of MSC in vitro and this indicates that our stem and progenitor cells undergo a similar process also in vivo

Glucosylceramide Synthase Is Involved in Development of Invariant Natural Killer T Cells

Invariant natural killer T (iNKT) cells represent a unique population of CD1d-restricted T lymphocytes expressing an invariant T cell receptor encoded by Vα14-Jα18 and Vα24-Jα18 gene segments in mice and humans, respectively. Recognition of CD1d-loaded endogenous lipid antigen(s) on CD4/CD8-double positive (DP) thymocytes is essential for the development of iNKT cells. The lipid repertoire of DP thymocytes and the identity of the decisive endogenous lipid ligands have not yet been fully elucidated. Glycosphingolipids (GSL) were implicated to serve as endogenous ligands. However, further in vivo investigations were hampered by early embryonal lethality of mice deficient for the key GSL-synthesizing enzyme glucosylceramide (GlcCer) synthase [GlcCer synthase (GCS), EC 2.4.1.80]. We have now analyzed the GSL composition of DP thymocytes and shown that GlcCer represented the sole neutral GSL and the acidic fraction was composed of gangliosides. Furthermore, we report on a mouse model that by combination of Vav-promoter-driven iCre and floxed GCS alleles (VavCreGCSf/f) enabled an efficient depletion of GCS-derived GSL very early in the T cell development, reaching a reduction by 99.6% in DP thymocytes. Although the general T cell population remained unaffected by this depletion, iNKT cells were reduced by approximately 50% in thymus, spleen, and liver and showed a reduced proliferation and an increased apoptosis rate. The Vβ-chains repertoire and development of iNKT cells remained unaltered. The GSL-depletion neither interfered with expression of CD1d, SLAM, and Ly108 molecules nor impeded the antigen presentation on DP thymocytes. These results indicate that GlcCer-derived GSL, in particular GlcCer, contribute to the homeostatic development of iNKT cells

Telomere-Dependent Chromosomal Instability

Telomeres are specialized DNA-protein structures at the ends of the linear chromosomes. In mammalian cells, they are composed of multifold hexameric TTAGGG repeats and a number of associated proteins. The double-stranded telomeric DNA ends in a 3′ single stranded overhang of 150 to 300 base pair (bp) which is believed to be required for a higher order structure (reviewed in (Blackburn, 2001)). One important model is that the telomeres form loop structures, the T-loops, and by invasion of the 3′ overhang into the duplex region of the double stranded part protect the DNA against degradation and hinder the cellular machinery to recognize the ends as broken DNA, thus providing chromosomal integrity (Griffith et al, 1999). If telomeres become critically short they loose their capping function, become sticky, and are prone to illegitimate chromosome end-to-end fusions. The resulting dicentric chromosomes are highly unusable and because of bridge-fusion-breakage cycles they give rise to chromosomal translocations, deletions, and amplifications. Thus, critically short telomeres are thought to be responsible for the onset of genomic instability. In addition, we provide evidence that in a length-independent manner telomeres can confer to genomic instability by forming telomeric aggregates which through chromosomal dys-locations contribute to chromosomal aberrations

Podoplanin is required for tumor cell invasion in cutaneous squamous cell carcinoma

The invasiveness of late-stage cutaneous squamous cell carcinoma (cSCC) is associated with poor patients' prognosis and linked to strong upregulation of the glycoprotein Podoplanin (PDPN) in cancer cells. However, the function of PDPN in these processes in cSCC carcinogenesis has not been characterized in detail yet. Employing a CRISPR/Cas9-based loss-of-function approach on murine cSCC cells, we show that the loss of Pdpn results in decreased migration and invasion in vitro. Complementing these in vitro studies, labelled murine control and Pdpn knockout cells were injected orthotopically into the dermis of nude mice to recapitulate the formation of human cSCC displaying a well-differentiated morphology with a PDPN-positive reaction in fibroblasts in the tumor stroma. Smaller tumors were observed upon Pdpn loss, which is associated with reduced tumor cell infiltration into the stroma. Utilizing Pdpn mutants in functional experiments in vitro, we provide evidence that both the intra- and extracellular domains are essential for cancer cell invasion. These findings underline the critical role of PDPN in cSCC progression and highlight potential therapeutic strategies targeting PDPN-dependent cancer cell invasion, especially in late-stage cSCC patients

CD73 Overexpression in Podocytes: A Novel Marker of Podocyte Injury in Human Kidney Disease

The CD73 pathway is an important anti-inflammatory mechanism in various disease settings. Observations in mouse models suggested that CD73 might have a protective role in kidney damage; however, no direct evidence of its role in human kidney disease has been described to date. Here, we hypothesized that podocyte injury in human kidney diseases alters CD73 expression that may facilitate the diagnosis of podocytopathies. We assessed the expression of CD73 and one of its functionally important targets, the C-C chemokine receptor type 2 (CCR2), in podocytes from kidney biopsies of 39 patients with podocytopathy (including focal segmental glomerulosclerosis (FSGS), minimal change disease (MCD), membranous glomerulonephritis (MGN) and amyloidosis) and a control group. Podocyte CD73 expression in each of the disease groups was significantly increased in comparison to controls (p < 0.001–p < 0.0001). Moreover, there was a marked negative correlation between CD73 and CCR2 expression, as confirmed by immunohistochemistry and immunofluorescence (Pearson r = −0.5068, p = 0.0031; Pearson r = −0.4705, p = 0.0313, respectively), thus suggesting a protective role of CD73 in kidney injury. Finally, we identify CD73 as a novel potential diagnostic marker of human podocytopathies, particularly of MCD that has been notorious for the lack of pathological features recognizable by light microscopy and immunohistochemistry

Broad-Spectrum HDAC Inhibitors Promote Autophagy through FOXO Transcription Factors in Neuroblastoma

Depending on context and tumor stage, deregulation of autophagy can either suppress tumorigenesis or promote chemoresistance and tumor survival. Histone deacetylases (HDACs) can modulate autophagy; however, the exact mechanisms are not fully understood. Here, we analyze the effects of the broad-spectrum HDAC inhibitors (HDACi) panobinostat and vorinostat on the transcriptional regulation of autophagy with respect to autophagy transcription factor activity (Transcription factor EB—TFEB, forkhead boxO—FOXO) and autophagic flux in neuroblastoma cells. In combination with the late-stage autophagic flux inhibitor bafilomycin A1, HDACis increase the number of autophagic vesicles, indicating an increase in autophagic flux. Both HDACi induce nuclear translocation of the transcription factors FOXO1 and FOXO3a, but not TFEB and promote the expression of pro-autophagic FOXO1/3a target genes. Moreover, FOXO1/3a knockdown experiments impaired HDACi treatment mediated expression of autophagy related genes. Combination of panobinostat with the lysosomal inhibitor chloroquine, which blocks autophagic flux, enhances neuroblastoma cell death in culture and hampers tumor growth in vivo in a neuroblastoma zebrafish xenograft model. In conclusion, our results indicate that pan-HDACi treatment induces autophagy in neuroblastoma at a transcriptional level. Combining HDACis with autophagy modulating drugs suppresses tumor growth of high-risk neuroblastoma cells. These experimental data provide novel insights for optimization of treatment strategies in neuroblastoma

Next generation multi-scale biophysical characterization of high precision cancer particle radiotherapy using clinical proton, helium-, carbon- and oxygen ion beams

The growing number of particle therapy facilities worldwide landmarks a novel era of precision oncology. Implementation of robust biophysical readouts is urgently needed to assess the efficacy of different radiation qualities. This is the first report on biophysical evaluation of Monte Carlo simulated predictive models of prescribed dose for four particle qualities i.e., proton, helium-, carbon- or oxygen ions using raster-scanning technology and clinical therapy settings at HIT. A high level of agreement was found between the in silico simulations, the physical dosimetry and the clonogenic tumor cell survival. The cell fluorescence ion track hybrid detector (Cell-Fit-HD) technology was employed to detect particle traverse per cell nucleus. Across a panel of radiobiological surrogates studied such as late ROS accumulation and apoptosis (caspase 3/7 activation), the relative biological effectiveness (RBE) chiefly correlated with the radiation species-specific spatio-temporal pattern of DNA double strand break (DSB) formation and repair kinetic. The size and the number of residual nuclear γ-H2AX foci increased as a function of linear energy transfer (LET) and RBE, reminiscent of enhanced DNA-damage complexity and accumulation of non-repairable DSB. These data confirm the high relevance of complex DSB formation as a central determinant of cell fate and reliable biological surrogates for cell survival/ RBE. The multi-scale simulation, physical and radiobiological characterization of novel clinical quality beams presented here constitutes a first step towards development of high precision biologically individualized radiotherapy