Discrimination of papillary thyroid cancer from non-cancerous thyroid tissue based on lipid profiling by mass spectrometry imaging

Introduction: The distinction of papillary thyroid carcinomas from benign thyroid lesions has important implication for clinical management. Classification based on histopathological features can be supported by molecular biomarkers, including lipidomic signatures, identified with the use of high-throughput mass spectrometry techniques. Formalin fixation is a standard procedure for stabilization and preservation of tissue samples, therefore this type of samples constitute highly valuable source of clinical material for retrospective molecular studies. In this study we used mass spectrometry imaging to detect lipids discriminating papillary cancer from not cancerous thyroid directly in formalin-fixed tissue sections. Material and methods: For this purpose imaging and profiling of lipids present in non-malignant and cancerous thyroid tissue specimens were conducted. High resolution MALDI-Q-Ion Mobility-TOF-MS technique was used for lipidomic analysis of formalin fixed thyroid tissue samples. Lipids were identified by the comparison of the exact molecular masses and fragmentation pathways of the protonated molecule ions, recorded during the MS/MS experiments, with LIPID MAPS database. Results: Several phosphatidylcholines (32:0, 32:1, 34:1 and 36:3), sphingomyelins (34:1 and 36:1) and phosphatidic acids (36:2 and 36:3) were detected and their abundances were significantly higher in cancerous tissue compared to non-cancerous tissue. The same lipid species were detected in formalin-fixed as in fresh-frozen tissue, but [M + Na]+ions were the most abundant in formalin fixed whereas [M + K]+ions were predominant in fresh tissue. Conclusions: Our results prove the viability of MALDI-MSI for analysis of lipid distribution directly in formalin-fixed tissue, and the potential for their use in the classification of thyroid diseases

Small DNA Pieces in C. elegans Are Intermediates of DNA Fragmentation during Apoptosis

While studying small noncoding RNA in C. elegans, we discovered that protocols used for isolation of RNA are contaminated with small DNA pieces. After electrophoresis on a denaturing gel, the DNA fragments appear as a ladder of bands, ∼10 nucleotides apart, mimicking the pattern of nuclease digestion of DNA wrapped around a nucleosome. Here we show that the small DNA pieces are products of the DNA fragmentation that occurs during apoptosis, and correspondingly, are absent in mutant strains incapable of apoptosis. In contrast, the small DNA pieces are present in strains defective for the engulfment process of apoptosis, suggesting they are produced in the dying cell prior to engulfment. While the small DNA pieces are also present in a number of strains with mutations in predicted nucleases, they are undetectable in strains containing mutations in nuc-1, which encodes a DNase II endonuclease. We find that the small DNA pieces can be labeled with terminal deoxynucleotidyltransferase only after phosphatase treatment, as expected if they are products of DNase II cleavage, which generates a 3′ phosphate. Our studies reveal a previously unknown intermediate in the process of apoptotic DNA fragmentation and thus bring us closer to defining this important pathway

TRAIL treatment provokes mutations in surviving cells

Chemotherapy and radiotherapy commonly damage DNA and trigger p53-dependent apoptosis through intrinsic apoptotic pathways. Two unfortunate consequences of this mechanism are resistance due to blockade of p53 or intrinsic apoptosis pathways, and mutagenesis of non-malignant surviving cells which can impair cellular function or provoke second malignancies. Death ligand-based drugs, such as tumor necrosis factor-related apoptosis inducing ligand (TRAIL), stimulate extrinsic apoptotic signaling, and may overcome resistance to treatments that induce intrinsic apoptosis. As death receptor ligation does not damage DNA as a primary mechanism of pro-apoptotic action, we hypothesized that surviving cells would remain genetically unscathed, suggesting that death ligand-based therapies may avoid some of the adverse effects associated with traditional cancer treatments. Surprisingly, however, treatment with sub-lethal concentrations of TRAIL or FasL was mutagenic. Mutations arose in viable cells that contained active caspases, and overexpression of the caspase-8 inhibitor crmA or silencing of caspase-8 abolished TRAIL-mediated mutagenesis. Downregulation of the apoptotic nuclease caspase-activated DNAse (CAD)/DNA fragmentation factor 40 (DFF40) prevented the DNA damage associated with TRAIL treatment. Although death ligands do not need to damage DNA in order to induce apoptosis, surviving cells nevertheless incur DNA damage after treatment with these agents

The Early Apoptotic DNA Fragmentation Targets a Small Number of Specific Open Chromatin Regions

We report here that early apoptotic DNA fragmentation, as obtained by using an entirely new approach, is the result of an attack at a small number of specific open chromatin regions of interphase nuclei. This was demonstrated as follows: (i) chicken liver was excised and kept in sterile tubes for 1 to 3 hours at 37°C; (ii) this induced apoptosis (possibly because of oxygen deprivation), as shown by the electrophoretic nucleosomal ladder produced by DNA preparations; (iii) low molecular-weight DNA fragments (∼200 bp) were cloned, sequenced, and shown to derive predominantly from genes and surrounding 100 kb regions; (iv) a few hundred cuts were produced, very often involving the same chromosomal sites; (v) at comparable DNA degradation levels, micrococcal nuclease (MNase) also showed a general preference for genes and surrounding regions, but MNase cuts were located at sites that were quite distinct from, and less specific than, those cut by apoptosis. In conclusion, the approach presented here, which is the mildest and least intrusive approach, identifies a preferred accessibility landscape in interphase chromatin

Seminiferous tubule transfection in vitro to define post-meiotic gene regulation

The electronic version of this article is the complete one and can be found online at: http://www.rbej.com/content/7/1/67Background: Post-meiotically expressed genes in the testis are essential for the proper progression of spermatogenesis, and yet, aside from the construction of individual transgenic mice using specific promoters to drive reporter plasmids, there are only very limited possibilities for relevant and quantitative analysis of gene promoters. This is due to the special nature of post-meiotic haploid cells, which to date are not represented in any appropriate cell-lines. This article reports the development of novel methodology using isolated and cultured rat seminiferous tubules in a multiwell format, into which promoter-reporter constructs can be introduced by a combination of microinjection and electroporation. Methods: Culture conditions were developed which allowed the continued incubation of isolated rat seminiferous tubules for up to 48 h without obvious cell death and loss of post-meiotic cells. Transfection of intact seminiferous tubules by microinjection and electroporation was optimized to achieve high expression efficiencies of control plasmids, using either fluorescent protein or luciferase as reporters, thereby allowing both morphological as well as quantitative assessment. Results: Successful transfection was achieved into all cell types except for mature spermatozoa. However, there appeared to be only limited cell-type specificity for the promoters used, even though these had appeared to be specific when used in transgenic animals. Conclusion: We have devised a methodology which allows relatively high throughput analysis of post-meiotic gene promoters into primary cells of intact seminiferous tubules. An apparent lack of cell-type specificity suggests that the gene fragments used do not contain sufficient targeting information, or that the transient episomal expression of the constructs does not encourage appropriate expression specificity. The results also highlight the doubtful interpretation of many studies using heterologous transfection systems to analyse post-meiotically expressed genes.Sandra Danner, Christiane Kirchhoff and Richard Ivel

Resistance to caspase-8 and -9 fragments in a malignant pleural mesothelioma cell line with acquired cisplatin-resistance

Apoptotic cysteine–aspartate proteases (caspases) are essential for the progression and execution of apoptosis, and detection of caspase fragmentation or activity is often used as markers of apoptosis. Cisplatin (cis-diamminedichloroplatinum (II)) is a chemotherapeutic drug that is clinically used for the treatment of solid tumours. We compared a cisplatin-resistant pleural malignant mesothelioma cell line (P31res1.2) with its parental cell line (P31) regarding the consequences of in vitro acquired cisplatin-resistance on basal and cisplatin-induced (equitoxic and equiapoptotic cisplatin concentrations) caspase-3, -8 and -9 fragmentation and proteolytic activity. Acquisition of cisplatin-resistance resulted in basal fragmentation of caspase-8 and -9 without a concomitant increase in proteolytic activity, and there was an increased basal caspase-3/7 activity. Similarly, cisplatin-resistant non-small-cell lung cancer cells, H1299res, had increased caspase-3 and -9 content compared with the parental H1299 cells. In P31 cells, cisplatin exposure resulted in caspase-9-mediated caspase-3/7 activation, but in P31res1.2 cells the cisplatin-induced caspase-3/7 activation occurred before caspase-8 or -9 activation. We therefore concluded that in vitro acquisition of cisplatin-resistance rendered P31res1.2 cells resistant to caspase-8 and caspase-9 fragments and that cisplatin-induced, initiator-caspase independent caspase-3/7 activation was necessary to overcome this resistance. Finally, the results demonstrated that detection of cleaved caspase fragments alone might be insufficient as a marker of caspase activity and ensuing apoptosis induction

Next-Generation Sequencing of Apoptotic DNA Breakpoints Reveals Association with Actively Transcribed Genes and Gene Translocations

DNA fragmentation is a well-recognized hallmark of apoptosis. However, the precise DNA sequences cleaved during apoptosis triggered by distinct mechanisms remain unclear. We used next-generation sequencing of DNA fragments generated in Actinomycin D-treated human HL-60 leukemic cells to generate a high-throughput, global map of apoptotic DNA breakpoints. These data highlighted that DNA breaks are non-random and show a significant association with active genes and open chromatin regions. We noted that transcription factor binding sites were also enriched within a fraction of the apoptotic breakpoints. Interestingly, extensive apoptotic cleavage was noted within genes that are frequently translocated in human cancers. We speculate that the non-random fragmentation of DNA during apoptosis may contribute to gene translocations and the development of human cancers

Synchronized turbo apoptosis induced by cold-shock

In our research on the role of apoptosis in the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE), we aim to evaluate the effects of early and late apoptotic cells and blebs on antigen presenting cells. This requires the in vitro generation of sufficiently large and homogeneous populations of early and late apoptotic cells. Here, we present a quick method encountered by serendipity that results in highly reproducible synchronized homogeneous apoptotic cell populations. In brief, granulocytic 32Dcl3 cells are incubated on ice for 2 h and subsequently rewarmed at 37°C. After 30–90 min at 37°C more than 80–90% of the cells become early apoptotic (Annexin V positive/propidium iodide negative). After 24 h of rewarming at 37°C 98% of the cells were late apoptotic (secondary necrotic; Annexin V positive/propidium iodide positive). Cells already formed apoptotic blebs at their cell surface after approximately 20 min at 37°C. Inter-nucleosomal chromatin cleavage and caspase activation were other characteristics of this cold-shock-induced process of apoptosis. Consequently, apoptosis could be inhibited by a caspase inhibitor. Finally, SLE-derived anti-chromatin autoantibodies showed a high affinity for apoptotic blebs generated by cold-shock. Overall, cold-shock induced apoptosis is achieved without the addition of toxic compounds or antibodies, and quickly leads to synchronized homogeneous apoptotic cell populations, which can be applied for various research questions addressing apoptosis

Contribution of histone sequence preferences to nucleosome organization: proposed definitions and methodology

We propose definitions and procedures for comparing nucleosome maps and discuss current agreement and disagreement on the effect of histone sequence preferences on nucleosome organization in vivo