Radiation Induced Lung Injury (RILI) is one of the main limiting factors of thorax irradiation, which can induce acute pneumonitis as well as pulmonary fibrosis, the latter being a life-threatening ...condition. The order of cellular and molecular events in the progression towards fibrosis is key to the physiopathogenesis of the disease, yet their coordination in space and time remains largely unexplored. Here, we present an interactive murine single cell atlas of the lung response to irradiation, generated from C57BL6/J female mice. This tool opens the door for exploration of the spatio-temporal dynamics of the mechanisms that lead to radiation-induced pulmonary fibrosis. It depicts with unprecedented detail cell type-specific radiation-induced responses associated with either lung regeneration or the failure thereof. A better understanding of the mechanisms leading to lung fibrosis will help finding new therapeutic options that could improve patients' quality of life.
To rapidly assess healthy tissue toxicities induced by new anti-cancer therapies (i.e., radiation alone or in combination with drugs), there is a critical need for relevant and easy-to-use models. ...Consistent with the ethical desire to reduce the use of animals in medical research, we propose to monitor lung toxicity using an ex vivo model. Briefly, freshly prepared organotypic lung slices from mice were irradiated, with or without being previously exposed to chemotherapy, and treatment toxicity was evaluated by analysis of cell division and viability of the slices. When exposed to different doses of radiation, this ex vivo model showed a dose-dependent decrease in cell division and viability. Interestingly, monitoring cell division was sensitive enough to detect a sparing effect induced by FLASH radiotherapy as well as the effect of combined treatment. Altogether, the organotypic lung slices can be used as a screening platform to rapidly determine in a quantitative manner the level of lung toxicity induced by different treatments alone or in combination with chemotherapy while drastically reducing the number of animals. Translated to human lung samples, this ex vivo assay could serve as an innovative method to investigate patients’ sensitivity to radiation and drugs.
Telomere length is an important parameter of telomere function since it determines number of aspects controlling chromosome stability and cell division. Since telomeres shorten with age in humans and ...premature aging syndromes are often associated with the presence of short telomeres, it has been proposed that telomere length is also an important parameter for organismal aging. How mean telomere lengths are determined in humans remains puzzling, but it is clear that genetic and epigenetic factors appear to be of great importance. Experimental evidence obtained from many different organisms has provided the basis for a widely accepted counting mechanism based on a negative feedback loop for telomerase activity at the level of individual telomeres. In addition, recent studies in both normal and pathological contexts point to the existence of chromosome-specific mechanisms of telomere length regulation determining a telomere length profile, which is inherited and maintained throughout life. In this review, we recapitulate the available data, propose a synthetic view of telomere length control mechanisms in humans and suggest new approaches to test current hypotheses.
Breast cancer is the most common malignancy among women. Current therapies for breast tumors are based on the use of chemotherapeutic drugs that are quite toxic for the patients and often result in ...resistance. Telomerase is up-regulated in 95% of breast carcinomas but not in adjacent normal tissues. Therefore, it represents a very promising target for anticancer therapies. Unfortunately, the antiproliferative effects of telomerase inhibition require extensive telomere shortening before they are fully present. Combining telomerase inhibition with common chemotherapeutic drugs can be used to reduce this lag phase and induce tumor cell death more effectively. Few studies have analyzed the effects of telomerase inhibition in combination with anticancer drugs in breast cancer cells. In this study, we inhibited telomerase activity in two breast cancer cell lines using a dominant-negative human telomerase reverse transcriptase and analyzed cell viability after treatment with different anticancer compounds. We found that dominant-negative human telomerase reverse transcriptase efficiently inhibits telomerase activity and causes telomere shortening over time. Moreover, cells in which telomerase was suppressed were more sensitive to anticancer agents independently of their mechanism of action and this sensitization was dependent on the presence of shorter telomeres. Altogether, our data show that blocking telomere length maintenance in combination with anticancer drugs can be used as an effective way to induce death of breast cancer cells.
Telomere maintenance activity is a hallmark of cancer. In some telomerase-negative tumors, telomeres become lengthened by alternative lengthening of telomeres (ALT), a recombination-mediated DNA ...replication process in which telomeres use other telomeric DNA as a copy template. Using chromosome orientation fluorescence in situ hybridization, we found that postreplicative exchange events involving a telomere and another TTAGGG-repeat tract occur at remarkably high frequencies in ALT cells (range 28-280/100 metaphases) and rarely or never in non-ALT cells, including cell lines with very long telomeres. Like the ALT phenotype itself, the telomeric exchanges were not suppressed when telomerase was activated in ALT cells. These exchanges are telomere specific because there was no correlation with sister chromatid exchange rates at interstitial locations, and they were not observed in non-ALT Bloom syndrome cells with very high sister chromatid exchange rates.
Telomere instability and cancer Londoño-Vallejo, J. Arturo
Biochimie,
2008, 2008-Jan, 2008-1-00, 20080101, Volume:
90, Issue:
1
Journal Article
Peer reviewed
Telomeres are required to preserve genome integrity, chromosome stability, nuclear architecture and chromosome pairing during meiosis. Given that telomerase activity is limiting or absent in most ...somatic tissues, shortening of telomeres during development and aging is the rule.
In vitro, telomere length operates as a mechanism to prevent uncontrolled cell growth and therefore defines the proliferation potential of a cell.
In vitro, in somatic cells that have lost proliferation control, shortening of telomeres becomes the main source of genome instability leading to genetic or epigenetic changes that may allow cells to become immortal and to acquire tumor phenotypes.
In vivo, mice models have indisputably shown both the protective and the promoting role of very short telomeres in cancer development. In humans, although telomere shortening and other types of telomere dysfunction probably contribute to the genome instability often detected in tumors, the specific contributions of such instability to the development of cancer remain largely undetermined.
The stability of the ends of linear eukaryotic chromosomes is ensured by functional telomeres, which are composed of short, species-specific direct repeat sequences. The maintenance of telomeres ...depends on a specialized ribonucleoprotein (RNP) called telomerase. Both telomeres and telomerase are dynamic entities with different physical behaviors and, given their substrate–enzyme relation, they must establish a productive interaction. Regulatory mechanisms controlling this interaction are key missing elements in our understanding of telomere functions. Here, we review the dynamic properties of telomeres and the maturing telomerase RNPs, and summarize how tracking the timing of their dance during the cell cycle will yield insights into chromosome stability mechanisms. Cancer cells often display loss of genome integrity; therefore, these issues are of particular interest for our understanding of cancer initiation or progression.
Guanine-rich nucleic acids can fold into the non-B DNA or RNA structures called G-quadruplexes (G4). Recent methodological developments have allowed the characterization of specific G-quadruplex ...structures in vitro as well as in vivo, and at a much higher throughput, in silico, which has greatly expanded our understanding of G4-associated functions. Typically, the consensus motif G3+N1-7G3+N1-7G3+N1-7G3+ has been used to identify potential G-quadruplexes from primary sequence. Since, various algorithms have been developed to predict the potential formation of quadruplexes directly from DNA or RNA sequences and the number of studies reporting genome-wide G4 exploration across species has rapidly increased. More recently, new methodologies have also appeared, proposing other estimates which consider non-canonical sequences and/or structure propensity and stability. The present review aims at providing an updated overview of the current open-source G-quadruplex prediction algorithms and straightforward examples of their implementation.
There is compelling evidence associating senescent cells with the malignant progression of tumours. Of all senescence-related mechanisms, the so-called senescence-associated secretory phenotype ...(SASP) has attracted much attention. Since the pro-inflammatory cytokines IL-6 and IL-8 are consistently present in the SASP, and secreted by highly aggressive breast cancer cell lines, we aimed at elucidating their role on the less aggressive breast cancer cell line MCF-7, which does not secret these cytokines.
The MCF-7 cell line was treated with either senescence-conditioned medium (SCM), IL-6 or IL-8 and then evaluated for phenotypic (CD44 and CD24 by FACS) and functional changes associated with an EMT program (migration/invasion) and for the acquisition of stem cell properties: mammosphere-forming capacity, expression of reprogramming factors (by qRT-PCR) and multilineage differentiation potential. We also evaluated the role of IL6 and IL8 in the cytokine-secreting, highly tumorigenic cell line MDA-MB-231.
Our results show that treatment of MCF-7 cells with IL6 and IL8, alone or together, induced the appearance of cells with fibroblastoid morphology, increased CD44 expression and migration, self-renewal and multilineage differentiation capacity, all characteristics compatible with an EMT program and stemness. These changes closely resembled those induced by a SCM. Interestingly, SCM treatments further increased IL6 and IL8 secretion by MCF-7 cells, thus suggesting the participation of an autocrine loop. Indeed, neutralizing antibodies against IL6 and IL8 reversed the effects of SCM on MCF-7, pinpointing these cytokines as major mediators of EMT and stemness-related effects associated with the senescent microenvironment. Additionally, prolonged exposure of MCF cells to IL6 or IL8 induced the appearance of senescent cells, suggesting a mechanism by which senescence and inflammation are reinforced favouring the acquisition of EMT and stem-like features at the population level, thus increasing tumour aggressiveness. Strikingly, our results also show that both IL6 and IL8 are important to maintain aggressive traits in MDA-MB-231 cells, a highly tumorigenic cell line, which appears to be devoid of stemness-related features.
This study demonstrates that, similar to what is observed with a senescent microenvironment, purified IL6 and IL8 induce a self- and cross-reinforced senescence/inflammatory milieu responsible for the emergence of epithelial plasticity and stemness features, thus conferring more aggressive phenotypes to a luminal breast cancer cell line. On the other hand, the basal-like MDA-MB-231 cells, whose aggressiveness-related features depend on IL6 and IL8 secretion, almost completely lack mammosphere formation and differentiation capacities, suggesting that tumour aggressiveness is not always related to stemness.
Epithelial‐to‐mesenchymal transition (EMT) describes the loss of epithelial traits and gain of mesenchymal traits by normal cells during development and by neoplastic cells during cancer metastasis. ...The long noncoding RNA HOTAIR triggers EMT, in part by serving as a scaffold for PRC2 and thus promoting repressive histone H3K27 methylation. In addition to PRC2, HOTAIR interacts with the LSD1 lysine demethylase, an epigenetic regulator of cell fate during development and differentiation, but little is known about the role of LSD1 in HOTAIR function during EMT. Here, we show that HOTAIR requires its LSD1‐interacting domain, but not its PRC2‐interacting domain, to promote the migration of epithelial cells. This activity is suppressed by LSD1 overexpression. LSD1‐HOTAIR interactions induce partial reprogramming of the epithelial transcriptome altering LSD1 distribution at promoter and enhancer regions. Thus, we uncover an unexpected role of HOTAIR in EMT as an LSD1 decommissioning factor, counteracting its activity in the control of epithelial identity.
SYNOPSIS
This study shows that HOTAIR lncRNA independently of PRC2 redistributes LSD1 along chromatin genome‐wide and thus impairs Lsd1 function in maintaining epithelial identity.
HOTAIR lncRNA promotes migration of immortalized epithelial cells.
The Lsd1‐binding domain, but not the PRC2‐binding domain, is essential for the HOTAIR‐dependent migration phenotype.
HOTAIR displaces Lsd1 from its target enhancers and promoters in epithelial cells.
Lsd1 dislocation in the presence of HOTAIR switches gene expression patterns to favor mesenchymal identity.
This study shows that HOTAIR lncRNA independently of PRC2 redistributes LSD1 along chromatin genome‐wide and thus impairs Lsd1 function in maintaining epithelial identity.
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