Effects of hTERT on genomic instability caused by either metals or radiation or combined exposure

There is currently a great interest in delayed chromosomal damage and other damaging effects of low-dose exposure to a variety of agents, which appear collectively to act through induction of stress-response pathways related to oxidative stress (and aging). These agents have been studied mostly in the radiation field but evidence is accumulating that chemicals, especially heavy metals, can also act in the same manner. Therefore, this work investigated the effects of metals and/or radiation in human fibroblasts in vitro. Humans are exposed to metals, including chromium (CR) VI) and vanadium (V) (V) from the environment, industry and surgical implants. Thus the impact of low-dose stress responses may be greater than expected from individual toxicity projections. In this study, a short (24 hours) exposure of human fibroblasts to low doses of Cr (VI) and V (V) caused both acute chromosome damage and genomic instability in the progeny of exposed cells for a t least 30 days after exposure. Acutely, Cr (VI) caused chromatid/ breaks without aneuploidy while V (V) caused aneuploidy without chromatid breaks. The long-term genomic instability was similar but depended on hTERT positivity. In telomerase-negative hTERT- cells, CR (VI) and V (V) caused a long lasting and transmissible induction of dicentric chromosome, nucleoplasmic bridges, micronuclei and aneuploidy. There was also a long term and transmissible reduction of clonogenic survival, with and increased b-galactosidase staining and apoptosis. This instability was not present in telomerase positive hTERT + cells. In contrast, in HTERT + cells the metals caused a persistent induction of tetraploidy, which was not noted in hTERT-cells. Interestingly, the clonogenic assay demonstrated that radiation induced genomic instability in hTERT + cells and to a lesser extent, in hTERT-cells. This showed that the telomerase activity in hTERT+cells did not provide protection against genomic instability caused by the radiation insult. Furthermore, neither 0.05 Gy nor 0.5 Gy doses of radiation induced chromosomal instability in either types of cells used (hTERT-and hTERT + cells). However, hTERT + cells had a slight higher incidence of micronuclei, immediately after radiation exposure of 0.5 Gy compared to hTERT- cells. Similarly to the metal only experiments, there was a higher level of tetraploidy in the hTERT+cells compared to the hTERT-cells, although it only reached a level of statistical significance immediately after the radiation exposure of the 0.05 Gy dose. This finding was different to what was seen for the metal only treated [CR (VI)] cells, where hTERT-cells showed significant cell damage and this damage was less compared to hTERT+cells. Combined exposure caused loss of clonogenic survival and therefore genomic instability in both types of cells (hTERT – and hTERT + cells). This genomic instability was more pronounced in hTERT + cells after Metal Followed by Radiation, and it was more pronounced in hTERT- cells after Radiation Followed by Metal. Similarly, cytogenetic damage was higher in hTERT+ cells after Metal Followed by Radiation, and higher in hTERT- cells after Radiation Followed by Metal. Similar to the metal only experiments, there was a higher level of tetraploidy in the hTERT +cells compared to the hTERT-cells, although it did not reach a level of statistical significance. It appears that the biological effects provoked by combined exposure of metal and radiation has led to a synergistic action in both types of cells, compared to metal treatment only or radiation exposure only. In fact, in most of the significant results, the damage caused by the combination of metal and radiation was higher than the damage induced by either metal itself or radiation itself. Similarly to the radiation only experiments, it was interesting to observe that ectopic hTERT expression had no effect in preventing the lost of clonogenic survival, as well as the formation of cell damage after combined exposure. This was in contrast to metal only treated [CR (VI)] cells, where hTERT – cells showed cell damage which was less compared to that observed in hTERT+cells. This study suggests that the type of genomic instability caused by metals in human cells may depend critically on whether they are telomerase-positive or – negative. However, the type of genomic instability caused by either radiation or combined exposure to metals and radiation in human cells appears to be not prevented by telomerase activity

Integrating multiple genome annotation databases improves the interpretation of microarray gene expression data

<p>Abstract</p> <p>Background</p> <p>The Affymetrix GeneChip is a widely used gene expression profiling platform. Since the chips were originally designed, the genome databases and gene definitions have been considerably updated. Thus, more accurate interpretation of microarray data requires parallel updating of the specificity of GeneChip probes. We propose a new probe remapping protocol, using the zebrafish GeneChips as an example, by removing nonspecific probes, and grouping the probes into transcript level probe sets using an integrated zebrafish genome annotation. This genome annotation is based on combining transcript information from multiple databases. This new remapping protocol, especially the new genome annotation, is shown here to be an important factor in improving the interpretation of gene expression microarray data.</p> <p>Results</p> <p>Transcript data from the RefSeq, GenBank and Ensembl databases were downloaded from the UCSC genome browser, and integrated to generate a combined zebrafish genome annotation. Affymetrix probes were filtered and remapped according to the new annotation. The influence of transcript collection and gene definition methods was tested using two microarray data sets. Compared to remapping using a single database, this new remapping protocol results in up to 20% more probes being retained in the remapping, leading to approximately 1,000 more genes being detected. The differentially expressed gene lists are consequently increased by up to 30%. We are also able to detect up to three times more alternative splicing events. A small number of the bioinformatics predictions were confirmed using real-time PCR validation.</p> <p>Conclusions</p> <p>By combining gene definitions from multiple databases, it is possible to greatly increase the numbers of genes and splice variants that can be detected in microarray gene expression experiments.</p

CHK1 inhibitor sensitizes resistant colorectal cancer stem cells to nortopsentin

Limited therapeutic options are available for advanced colorectal cancer (CRC). Herein, we report that exposure to a neo-synthetic bis(indolyl)thiazole alkaloid analog, nortopsentin 234 (NORA234), leads to an initial reduction of proliferative and clonogenic potential of CRC sphere cells (CR-CSphCs), followed by an adaptive response selecting the CR-CSphC-resistant compartment. Cells spared by the treatment with NORA234 express high levels of CD44v6, associated with a constitutive activation of Wnt pathway. In CR-CSphC-based organoids, NORA234 causes a genotoxic stress paralleled by G2-M cell cycle arrest and activation of CHK1, driving the DNA damage repair of CR-CSphCs, regardless of the mutational background, microsatellite stability, and consensus molecular subtype. Synergistic combination of NORA234 and CHK1 (rabusertib) targeting is synthetic lethal inducing death of both CD44v6-negative and CD44v6-positive CRC stem cell fractions, aside from Wnt pathway activity. These data could provide a rational basis to develop an effective strategy for the treatment of patients with CRC

Mechanisms of sensitivity and resistance to CDK4/CDK6 inhibitors in hormone receptor-positive breast cancer treatment

Cell cycle dysregulation is a hallmark of cancer that promotes eccessive cell division. Cyclin-dependent kinase 4 (CDK4) and cyclin-dependent kinase 6 (CDK6) are key molecules in the G1-to-S phase cell cycle transition and are crucial for the onset, survival, and progression of breast cancer (BC). Small-molecule CDK4/CDK6 inhibitors (CDK4/6i) block phosphorylation of tumor suppressor Rb and thus restrain susceptible BC cells in G1 phase. Three CDK4/6i are approved for the first-line treatment of patients with advanced/metastatic hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2-) BC in combination with endocrine therapy (ET). Though this has improved the clinical outcomes for survival of BC patients, there is no established standard next-line treatment to tackle drug resistance. Recent studies suggest that CDK4/6i can modulate other distinct effects in both BC and breast stromal compartments, which may provide new insights into aspects of their clinical activity. This review describes the biochemistry of the CDK4/6-Rb-E2F pathway in HR+ BC, then discusses how CDK4/6i can trigger other effects in BC/breast stromal compartments, and finally outlines the mechanisms of CDK4/6i resistance that have emerged in recent preclinical studies and clinical cohorts, emphasizing the impact of these findings on novel therapeutic opportunities in BC

PI3K/AKT/mTOR signaling transduction pathway and targeted therapies in cancer

Abstract The PI3K/AKT/mTOR (PAM) signaling pathway is a highly conserved signal transduction network in eukaryotic cells that promotes cell survival, cell growth, and cell cycle progression. Growth factor signalling to transcription factors in the PAM axis is highly regulated by multiple cross-interactions with several other signaling pathways, and dysregulation of signal transduction can predispose to cancer development. The PAM axis is the most frequently activated signaling pathway in human cancer and is often implicated in resistance to anticancer therapies. Dysfunction of components of this pathway such as hyperactivity of PI3K, loss of function of PTEN, and gain-of-function of AKT, are notorious drivers of treatment resistance and disease progression in cancer. In this review we highlight the major dysregulations in the PAM signaling pathway in cancer, and discuss the results of PI3K, AKT and mTOR inhibitors as monotherapy and in co-administation with other antineoplastic agents in clinical trials as a strategy for overcoming treatment resistance. Finally, the major mechanisms of resistance to PAM signaling targeted therapies, including PAM signaling in immunology and immunotherapies are also discussed.

Mechanisms of sensitivity and resistance to CDK4/CDK6 inhibitors in hormone receptor-positive breast cancer treatment

CDK4/6i resistance; Endocrine therapy; Breast cancerResistencia a CDK4/6i; Terapia endocrina; Cáncer de mamaResistència a CDK4/6i; Teràpia endocrina; Càncer de mamaCell cycle dysregulation is a hallmark of cancer that promotes eccessive cell division. Cyclin-dependent kinase 4 (CDK4) and cyclin-dependent kinase 6 (CDK6) are key molecules in the G1-to-S phase cell cycle transition and are crucial for the onset, survival, and progression of breast cancer (BC). Small-molecule CDK4/CDK6 inhibitors (CDK4/6i) block phosphorylation of tumor suppressor Rb and thus restrain susceptible BC cells in G1 phase. Three CDK4/6i are approved for the first-line treatment of patients with advanced/metastatic hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2-) BC in combination with endocrine therapy (ET). Though this has improved the clinical outcomes for survival of BC patients, there is no established standard next-line treatment to tackle drug resistance. Recent studies suggest that CDK4/6i can modulate other distinct effects in both BC and breast stromal compartments, which may provide new insights into aspects of their clinical activity. This review describes the biochemistry of the CDK4/6-Rb-E2F pathway in HR+ BC, then discusses how CDK4/6i can trigger other effects in BC/breast stromal compartments, and finally outlines the mechanisms of CDK4/6i resistance that have emerged in recent preclinical studies and clinical cohorts, emphasizing the impact of these findings on novel therapeutic opportunities in BC.A.P.K. was supported by a grant from the Singapore Ministry of Education (MOE-T2EP30120–0016)

Distribution of Toxigenic <i>Halomicronema</i> spp. in Adjacent Environments on the Island of Ischia: Comparison of Strains from Thermal Waters and Free Living in <i>Posidonia Oceanica</i> Meadows

Organisms adaptable to extreme conditions share the ability to establish protective biofilms or secrete defence toxins. The extracellular substances that are secreted may contain monosaccharides and other toxic compounds, but environmental conditions influence biofilm characteristics. Microorganisms that are present in the same environment achieve similar compositions, regardless of their phylogenetic relationships. Alternatively, cyanobacteria phylogenetically related may live in different environments, but we ignore if their physiological answers may be similar. To test this hypothesis, two strains of cyanobacteria that were both ascribed to the genus Halomicronema were isolated. H. metazoicum was isolated in marine waters off the island of Ischia (Bay of Naples, Italy), free living on leaves of Posidonia oceanica. Halomicronema sp. was isolated in adjacent thermal waters. Thus, two congeneric species adapted to different environments but diffused in the same area were polyphasically characterized by microscopy, molecular, and toxicity analyses. A variable pattern of toxicity was exhibited, in accordance with the constraints imposed by the host environments. Cyanobacteria adapted to extreme environments of thermal waters face a few competitors and exhibit a low toxicity; in contrast, congeneric strains that have adapted to stable and complex environments as seagrass meadows compete with several organisms for space and resources, and they produce toxic compounds that are constitutively secreted in the surrounding waters

A method for isolation of cone photoreceptors from adult zebrafish retinae

Background: Cone photoreceptors are specialised sensory retinal neurons responsible for photopic vision, colour perception and visual acuity. Retinal degenerative diseases are a heterogeneous group of eye diseases in which the most severe vision loss typically arises from cone photoreceptor dysfunction or degeneration. Establishing a method to purify cone photoreceptors from retinal tissue can accelerate the identification of key molecular determinants that underlie cone photoreceptor development, survival and function. The work herein describes a new method to purify enhanced green fluorescent protein (EGFP)-labelled cone photoreceptors from adult retina of Tg(3.2gnat2:EGFP) zebrafish. Results: Methods for dissecting adult zebrafish retinae, cell dissociation, cell sorting, RNA isolation and RNA quality control were optimised. The dissociation protocol, carried out with ~30 retinae from adult zebrafish, yielded approximately 6 × 106 cells. Flow cytometry cell sorting subsequently distinguished 1 × 106 EGFP+ cells and 4 × 106 EGFP− cells. Electropherograms confirmed downstream isolation of high-quality RNA with RNA integrity number (RIN) >7.6 and RNA concentration >5.7 ng/µl obtained from both populations. Reverse Transcriptase-PCR confirmed that the EGFP-positive cell populations express known genetic markers of cone photoreceptors that were not expressed in the EGFP-negative cell population whereas a rod opsin amplicon was only detected in the EGFP-negative retinal cell population. Conclusions: This work describes a valuable adult zebrafish cone photoreceptor isolation methodology enabling future identification of cone photoreceptor-enriched genes, proteins and signalling networks responsible for their development, survival and function. In addition, this advancement facilitates the identification of novel candidate genes for inherited human blindness.Bright Focus FoundationMacular Degeneration GrantWelcome TrustNational Institutes of Healt