Super-telomeres in transformed human fibroblasts

Telomere length maintenance is critical for organisms' long-term survival and cancer cell proliferation. Telomeres are kept within species-specific length ranges by the interplay between telomerase activity and telomeric chromatin organization. In this paper, we exploited telomerase immortalized human fibroblasts (cen3tel) that gradually underwent neoplastic transformation during culture propagation to study telomere composition and length regulation during the transformation process. Just after telomerase catalytic subunit (hTERT) expression, cen3tel telomeres shortened despite the presence of telomerase activity. At a later stage and concomitantly with transformation, cells started elongating telomeres, which reached a mean length greater than 100kb in about 900 population doublings. Super-telomeres were stable and compatible with cell growth and tumorigenesis. Telomere extension was associated with increasing levels of telomerase activity that were linked to the deregulation of endogenous telomerase RNA (hTERC) and exogenous telomerase reverse transcriptase (hTERT) expression. Notably, the increase in hTERC levels paralleled the increase in telomerase activity, suggesting that this subunit plays a role in regulating enzyme activity. Telomeres ranging in length between 10 and more than 100kb were maintained in an extendible state although TRF1 and TRF2 binding increased with telomere length. Super-telomeres neither influenced subtelomeric region global methylation nor the expression of the subtelomeric gene FRG1, attesting the lack of a clear-cut relationship between telomere length, subtelomeric DNA methylation and expression in human cells. The cellular levels of the telomeric proteins hTERT, TRF1, TRF2 and Hsp90 rose with transformation and were independent of telomere length, pointing to a role of these proteins in tumorigenesis

First report of Bufo bufo (Linnaeus, 1758) from Sardinia (Italy)

The Common toad Bufo bufo (Linneaus, 1758) was found for the first time in Sardinia in 2016 during herpetological surveys. The species appears to be well established in the finding area. Many adults and juveniles, tadpoles at different developmental stages, and eggs have been found during repeated monitoring. In order to infer the geographic origin of the Sardinian population, we amplified two mitochondrial markers (16S, cytb) and compared sequences with those available for the species across its natural range. We also screened samples for the presence of Bd pathogen to assess the risk of infection mediated by the species in the area. Results suggest that Sardinian individuals are genetically close to the Central Italian populations, although they show a unique distinct haplotype. Though the species should be considered allochthonous to the Island, further molecular and ecological data are urgently needed to assess the genetic structure and the possible impact on the local fauna, which is largely composed by endemic taxa. Particularly, possible interactions with other native amphibians like the green toad Bufo balearicus (Boettger, 1880), also present in the area, should be investigated, both in terms of competition for breeding sites and genetic pollution, as these species are already known to hybridize in the wild

Towards a Radio-guided Surgery with β−\beta^{-} Decays: Uptake of a somatostatin analogue (DOTATOC) in Meningioma and High Grade Glioma

A novel radio guided surgery (RGS) technique for cerebral tumors using $\beta^{-}$ radiation is being developed. Checking the availability of a radio-tracer that can deliver a $\beta^{-}$ emitter to the tumor is a fundamental step in the deployment of such technique. This paper reports a study of the uptake of 90Y labeled (DOTATOC) in the meningioma and the high grade glioma (HGG) and a feasibility study of the RGS technique in these cases.Comment: 21 pages, 5 figure

Reduced Expression of the ROCK Inhibitor Rnd3 Is Associated with Increased Invasiveness and Metastatic Potential in Mesenchymal Tumor Cells

BACKGROUND: Mesenchymal and amoeboid movements are two important mechanisms adopted by cancer cells to invade the surrounding environment. Mesenchymal movement depends on extracellular matrix protease activity, amoeboid movement on the RhoA-dependent kinase ROCK. Cancer cells can switch from one mechanism to the other in response to different stimuli, limiting the efficacy of antimetastatic therapies. METHODOLOGY AND PRINCIPAL FINDINGS: We investigated the acquisition and molecular regulation of the invasion capacity of neoplastically transformed human fibroblasts, which were able to induce sarcomas and metastases when injected into immunocompromised mice. We found that neoplastic transformation was associated with a change in cell morphology (from fibroblastic to polygonal), a reorganization of the actin cytoskeleton, a decrease in the expression of several matrix metalloproteases and increases in cell motility and invasiveness. In a three-dimensional environment, sarcomagenic cells showed a spherical morphology with cortical actin rings, suggesting a switch from mesenchymal to amoeboid movement. Accordingly, cell invasion decreased after treatment with the ROCK inhibitor Y27632, but not with the matrix protease inhibitor Ro 28-2653. The increased invasiveness of tumorigenic cells was associated with reduced expression of Rnd3 (also known as RhoE), a cellular inhibitor of ROCK. Indeed, ectopic Rnd3 expression reduced their invasive ability in vitro and their metastatic potential in vivo. CONCLUSIONS: These results indicate that, during neoplastic transformation, cells of mesenchymal origin can switch from a mesenchymal mode of movement to an amoeboid one. In addition, they point to Rnd3 as a possible regulator of mesenchymal tumor cell invasion and to ROCK as a potential therapeutic target for sarcomas

Transcription of Satellite III non-coding RNAs is a general stress response in human cells

In heat-shocked human cells, heat shock factor 1 activates transcription of tandem arrays of repetitive Satellite III (SatIII) DNA in pericentromeric heterochromatin. Satellite III RNAs remain associated with sites of transcription in nuclear stress bodies (nSBs). Here we use real-time RT-PCR to study the expression of these genomic regions. Transcription is highly asymmetrical and most of the transcripts contain the G-rich strand of the repeat. A low level of G-rich RNAs is detectable in unstressed cells and a 104-fold induction occurs after heat shock. G-rich RNAs are induced by a wide range of stress treatments including heavy metals, UV-C, oxidative and hyper-osmotic stress. Differences exist among stressing agents both for the kinetics and the extent of induction (>100- to 80.000-fold). In all cases, G-rich transcripts are associated with nSBs. On the contrary, C-rich transcripts are almost undetectable in unstressed cells and modestly increase after stress. Production of SatIII RNAs after hyper-osmotic stress depends on the Tonicity Element Binding Protein indicating that activation of the arrays is triggered by different transcription factors. This is the first example of a non-coding RNA whose transcription is controlled by different transcription factors under different growth conditions

Drug Treatment of Cancer Cell Lines: A Way to Select for Cancer Stem Cells?

Tumors are generally composed of different cell types. In recent years, it has been shown that in many types of cancers a subset of cells show peculiar characteristics, such as the ability to induce tumors when engrafted into host animals, self-renew and being immortal, and give rise to a differentiated progeny. These cells have been defined as cancer stem cells (CSCs) or tumor initiating cells. CSCs can be isolated both from tumor specimens and established cancer cell lines on the basis of their ability to exclude fluorescent dyes, express specific cell surface markers or grow in particular culture conditions. A key feature of CSCs is their resistance to chemotherapeutic agents, which could contribute to the remaining of residual cancer cells after therapeutic treatments. It has been shown that CSC-like cells can be isolated after drug treatment of cancer cell lines; in this review, we will describe the strategies so far applied to identify and isolate CSCs. Furthermore, we will discuss the possible use of these selected populations to investigate CSC biology and develop new anticancer drugs

Structural and Functional Characterization of Noncoding Repetitive RNAs Transcribed in Stressed Human Cells

Thermal and chemical stresses induce the formation in human cells of novel and transient nuclear structures called nuclear stress bodies (nSBs). These contain heat shock factor 1 (HSF-1) and a specific subset of pre-mRNA processing factors. Nuclear stress bodies are assembled on specific pericentromeric heterochromatic domains containing satellite III (SatIII) DNA. In response to stress, these domains change their epigenetic status from heterochromatin to euchromatin and are transcribed in poly-adenylated RNAs that remain associated with nSBs. In this article, we describe the cloning, sequencing, and functional characterization of these transcripts. They are composed of SatIII repeats and originate from the transcription of multiple sites within the SatIII arrays. Interestingly, the level of SatIII RNAs can be down-regulated both by antisense oligonucleotides and small interfering RNAs (siRNA). Knockdown of SatIII RNA by siRNAs requires the activity of Argonaute 2, a component of the RNA-induced silencing complex. Down-regulation of satellite III RNAs significantly affects the recruitment of RNA processing factors to nSBs without altering the association of HSF-1 with these structures nor the presence of acetylated histones within nSBs. Thus, satellite III RNAs have a major role in the formation of nSBs

First report of bufo bufo (Linnaeus, 1758) from sardinia (Italy)

The Common toad Bufo bufo (Linneaus, 1758) was found for the first time in Sardinia in 2016 during her-petological surveys. The species appears to be well established in the finding area. Many adults and juveniles, tadpoles at different developmental stages, and eggs have been found during repeated monitoring. In order to infer the geographic origin of the Sardinian population, we amplified two mitochondrial markers (16S, cytb) and compared sequences with those available for the species across its natural range. We also screened samples for the presence of Bd pathogen to assess the risk of infection mediated by the species in the area. Results suggest that Sardinian individuals are genetically close to the Central Italian populations, although they show a unique distinct haplotype. Though the species should be considered allochthonous to the Island, further molecular and ecological data are urgently needed to assess the genetic structure and the possible impact on the local fauna, which is largely composed by endemic taxa. Particularly, possible interactions with other native amphibians like the green toad Bufo balearicus (Boettger, 1880), also present in the area, should be investigated, both in terms of competition for breeding sites and genetic pollution, as these species are already known to hybridize in the wild