Mitotic catastrophe and endomitosis in tumour cells: an evolutionary key to a molecular solution

Following genotoxic insult, p53 mutated tumour cells undergo mitotic catastrophe. This is characterised by a switch from mitosis to the endocycle. The essential difference between mitosis and the endocycle is that in the latter, DNA synthesis is uncoupled from cell division, which leads to the formation of endopolyploid cells. Recent data suggests that a return from the endocycle into mitosis is also possible. Furthermore, our observations indicate that a particular type of endocycle known as endomitosis may be involved in this return. Here we review the role of endomitosis in the somatic reduction of polyploidy during development and its postulated role in the evolution of meiosis. Finally, we incorporate these evolutionary data to help interpret our most recent observations in the tumour cell system, which indicate a role for endomitosis and meiotic regulators, in particular p39mos in the segregation of genomes (somatic reduction) of these endopolyploid cells.     

Challenges in assessing pathogenicity based on frequency of variants in mismatch repair genes: an extreme case of a MSH2 variant and a meta-analysis

The clinical interpretation of variants in mismatch repair (MMR) genes associated with Lynch syndrome can be confusing when the functional nature of the variant is not clearly defined. We report an extreme case where a polymorphism in the MSH2 gene which had a low minor allele frequency, was misclassified as a mutation based on low evidential methods in the database and previous publications. We expanded this experience to perform a systematic meta-analysis in order to investigate other variants that have potentially been misclassified. Our results suggested that the interpretation of pathogenicity should be more cautious and emphasized the need for solid validation through multiple analyses including functional analysis for variants in MMR genes.     

DNA adducts in relation to lung tumour outcome are not markers of susceptibility following a single dose treatment of SWR, BALB/c and C57BL/6J mice with N-nitrosodiethylamine

The formation of DNA adducts by the covalent binding of genotoxic chemicals to DNA represents a valuable marker for assessing exposure to carcinogens but as yet the role of DNA adducts as a biomarker of carcinogenic susceptibility still needs to be clearly ascertained. To address this question an animal study was instigated using mice (SWR (high), BALB/c (intermediate) and C57BL/6J (low)) varying in their susceptibility to lung carcinogenesis. Groups of animals from each strain were dosed with a single intraperitoneal injection of saline or N -nitrosodiethylamine (NDEA) at 15 or 90 mg kg^-1 body weight. Lung and liver tissues were removed at different time points following dosing. Further groups of mice dosed with the same regime had urine samples collected 24 h post dosing and were then left up to 18 months to allow for the development of tumours. Immunoslot-blot analysis was used for the determination of N-7 ethylguanine (N-7EtG) and O⁶ ethylguanine (O⁶EtG) adduct levels in the DNA from the tissues and gas chromatography-mass spectrometry (GC-MS) was used to determine N-3 ethyladenine (N-3EtA) adduct levels in the urine samples. Levels of alkyltransferase (ATase) were also determined in the tissues. The results showed that the DNA adduct levels and persistence were similar across the three strains of mice following dosing with 15 and 90 mg kg^-1 NDEA. High levels of adducts were observed in the urine of the BALB/c strain, implying an increased metabolic or repair capacity in this strain. However there were no differences in the levels of ATase in the lung and liver of the three strains of mice following dosing with 15 mg kg^-1 NDEA. The incidence of tumours in C57BL/6J mice was lower compared with the other two strains and showed a dose dependent increase. The results from this study show that the differences in susceptibility to lung carcinogenesis between the three strains of mice do not appear to be linked to the formation of the two adducts detected. These results imply that dosing with NDEA resulted in toxicity which may have led to cell death and induction of tumours by compensatory cell proliferation. Although these results do not allow decisive conclusions to be drawn concerning the relationship between total levels of DNA adducts and differences in carcinogenic susceptibility for the three strains of mice it is clear that the increased presence of a DNA adduct in the target tissue increases the likelihood of tumour development.     

Stereotactic radiation therapy for skull base recurrences: Is a salvage approach still possible?

AimA literature review was performed to analyse the role of stereotactic radiotherapy given in a single shot or in a fractionated fashion for recurrent skull base tumours in order to ascertain if it can be a real salvage approach.BackgroundThe management of recurrent skull base tumours can have a curative or palliative intent and mainly includes surgery and RT.Materials and methodsOne-thousand-ninety-one articles were found in the search databases and the most relevant of them were analysed and briefly described.ResultsData on recurrences of meningioma, pituitary adenoma, craniopharyngioma, chordoma and chondrosarcoma, vestibular schwannoma, glomus jugulare tumours, olfactory neuroblastoma and recurrences from head and neck tumours invading the base of skull are reported highlighting the most relevant results in terms of local control, survival, side effects and complications.ConclusionsIn conclusion, it emerges that SRS and FSRT are effective and safe radiation modalities of realize real salvage treatment for recurrent skull base tumours.     

DNA adducts in relation to lung tumour outcome are not markers of susceptibility following a single dose treatment of SWR,BALB/c and C57BL/6J mice with N-nitrosodiethylamine

The formation of DNA adducts by the covalent binding of genotoxic chemicals to DNA represents a valuable marker for assessing exposure to carcinogens but as yet the role of DNA adducts as a biomarker of carcinogenic susceptibility still needs to be clearly ascertained. To address this question an animal study was instigated using mice (SWR (high), BALB/c (intermediate) and C57BL/6J (low)) varying in their susceptibility to lung carcinogenesis. Groups of animals from each strain were dosed with a single intraperitoneal injection of saline or N -nitrosodiethylamine (NDEA) at 15 or 90 mg kg^-1 body weight. Lung and liver tissues were removed at different time points following dosing. Further groups of mice dosed with the same regime had urine samples collected 24 h post dosing and were then left up to 18 months to allow for the development of tumours. Immunoslot-blot analysis was used for the determination of N-7 ethylguanine (N-7EtG) and O⁶ ethylguanine (O⁶EtG) adduct levels in the DNA from the tissues and gas chromatography-mass spectrometry (GC-MS) was used to determine N-3 ethyladenine (N-3EtA) adduct levels in the urine samples. Levels of alkyltransferase (ATase) were also determined in the tissues. The results showed that the DNA adduct levels and persistence were similar across the three strains of mice following dosing with 15 and 90 mg kg^-1 NDEA. High levels of adducts were observed in the urine of the BALB/c strain, implying an increased metabolic or repair capacity in this strain. However there were no differences in the levels of ATase in the lung and liver of the three strains of mice following dosing with 15 mg kg^-1 NDEA. The incidence of tumours in C57BL/6J mice was lower compared with the other two strains and showed a dose dependent increase. The results from this study show that the differences in susceptibility to lung carcinogenesis between the three strains of mice do not appear to be linked to the formation of the two adducts detected. These results imply that dosing with NDEA resulted in toxicity which may have led to cell death and induction of tumours by compensatory cell proliferation. Although these results do not allow decisive conclusions to be drawn concerning the relationship between total levels of DNA adducts and differences in carcinogenic susceptibility for the three strains of mice it is clear that the increased presence of a DNA adduct in the target tissue increases the likelihood of tumour development.     

Endopolyploidy in irradiated p53-deficient tumour cell lines: persistence of cell division activity in giant cells expressing Aurora-B kinase

Recent findings including computerised live imaging suggest that polyploidy cells transiently emerging after severe genotoxic stress (and named 'endopolyploid cells') may have a role in tumour regrowth after anti-cancer treatment. Until now, mostly the factors enabling metaphase were studied in them. Here we investigate the mitotic activities and the role of Aurora-B, in view of potential depolyploidisation of these cells, because Aurora-B kinase is responsible for coordination and completion of mitosis. We observed that endopolyploid giant cells are formed via different means in irradiated p53 tumours, by: (1) division/fusion of daughter cells creating early multi-nucleated cells; (2) asynchronous division/fusion of sub-nuclei of these multi-nucleated cells; (3) a series of polyploidising mitoses reverting replicative interphase from aborted metaphase and forming giant cells with a single nucleus; (4) micronucleation of arrested metaphases enclosing genome fragments; or (5) incomplete division in the multi-polar mitoses forming late multi-nucleated giant cells. We also observed that these activities can release para-diploid cells, although infrequently. While apoptosis typically occurs after a substantial delay in these cells, we also found that approximately 2% of the endopolyploid cells evade apoptosis and senescence arrest and continue some form of mitotic activity. We describe here that catalytically active Aurora-B kinase is expressed in the nuclei of many endopolyploid cells in interphase, as well as being present at the centromeres, mitotic spindle and cleavage furrow during their attempted mitotes. The totally micronucleated giant cells (containing sub-genomic fragments in multiple micronuclei) represented only the minor fraction which failed to undergo mitosis, and Aurora-B was absent from it. These observations suggest that most endopolyploid tumour cells are not reproductively inert and that Aurora-B may contribute to the establishment of resistant tumours post-irradiation.     

Peptidylglycine α-amidating monooxygenase (PAM) immunoreactivity and messenger RNA in human pituitary and increased expression in pituitary tumours

Bioactivity of many peptides depends upon post-translational -amidation of inactive precursors by two enzyme activities known collectively as peptidylglycine -amidating monooxygenase (PAM). PAM enzymes are particularly abundant in the pituitary. The distribution of PAM immunoreactivity and messenger ribonucleic acid (mRNA) in the adult human pituitary and in pituitary tumours was investigated by use of immunocytochemistry and in situ hybridisation. Immunoreactivity was present in numerous cells of the anterior lobe: staining was intense in a proportion of gonadotrophs and folliculo-stellate cells, but weaker in the majority of somatotrophs and lactotrophs, a few corticotrophs and occasional thyrotrophs. PAM staining was also present in nerves, pituicytes and some endocrine cells within the posterior lobe (the human intermediate zone). Forty pituitary tumours of various types were immunoreactive for PAM; more intensely and uniformly stained than normal anterior lobe. In situ hybridisation with digoxigenin-labelled probes demonstrated intense labelling for PAM mRNA in numerous cells in normal anterior pituitary and in tumours. Many regulatory peptides that require amidation for activity, potential targets for PAM, are present in the pituitary. Many tumour growth factors also require amidation and PAM may regulate these mitogenic peptides in tumours.     

Neoplasia in the Siberian weasel (<Emphasis Type="Italic">Mustela sibirica</Emphasis>): two case reports of fibrosarcoma and interstitial cell tumour

Two cases of neoplasia occurred in two adult Siberian weasels (Mustela sibirica) in a game reserve in southern Lower Saxony, Germany. The first case, a male weasel, showed an enlargement of the right testicle, which was surgically removed. The tumour was classified as an interstitial cell tumour. One year after surgery, the male weasel died by accident and revealed tumour lesions of uncertain histogenesis within the abdominal cavity and the spleen at necropsy. The second case, a female animal, became suspicious by a dermal mass in the upper left hind limb, which was also surgically excised. Pathological examinations revealed a fibrosarcoma that recurred twice after removal of the original mass. These two cases of neoplasia in the Siberian weasel are demonstrated with regard to clinical aspects and discussed on the basis of their pathomorphological features.     

Agrobacterium vaccinii sp. nov. isolated from galls on blueberry plants (Vaccinium corymbosum)

Four non-pathogenic strains isolated from the galls on blueberry plants (Vaccinium corymbosum) were characterized by using polyphasic taxonomic methods. Based on 16S rRNA gene phylogeny, strains were clustered within the genus Agrobacterium. Furthermore, multilocus sequence analysis (MLSA) based on the partial sequences of atpD, recA and rpoB housekeeping genes and whole-genome-based phylogeny indicated that the strains studied form a novel Agrobacterium species. Analyses showed that the strains belong to “rubi” sub-clade of Agrobacterium genus and their closest relatives are Agrobacterium rubi and “Agrobacterium bohemicum”. Average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) comparisons between genome sequences of representative strains B7.6^T and B19.1.4, and their closest relatives, confirmed the distinct phylogenetic position of studied strains, because obtained values were considerably below the proposed thresholds for the species delineation. The four strains studied were phenotypically distinguishable from other species of the genus Agrobacterium. Overall, polyphasic characterization showed that the strains studied represent a novel species of the genus Agrobacterium, for which the name Agrobacterium vaccinii sp. nov. is proposed. The type strain of A. vaccinii is B7.6^T (=CFBP 8740^T = LMG 31849^T).