Abstract
Background
Development of in vitro models of pediatric brain tumors (pBT) is instrumental for both understanding the contributing oncogenic molecular mechanisms and identifying and testing ...new therapeutic strategies. Primary cell lines should be established and managed to prevent epigenetic and genetic alterations and thus recapitulating the original tumor. DNA methylation (DM) is a stable epigenetic modification, altered in cancer and recently used to classify tumors. We aim to apply DM and Copy Number Variation (CNV) profiling to characterize pBT primary cell lines and tumors.
Methods
We investigated 34 pBT tissues from different histology paired to 52 their derived primary cultures in both 2D and 3D conditions, as stem-cells or in serum-supplemented medium, and both short and long-terms in culture. We studied 18 additional pBT-derived cell-lines, 9 organoids, 5 commercial cell-lines, and 122 pBT tissues from the same histological categories, as controls, for a total of 240 genome-wide DM profiles. We analyzed DM and CNV profiles by using Illumina EPIC-arrays. By means of a bump hunting strategy, we identified differentially methylated regions in faithful vs unfaithful cell lines, and performed a functional characterization using over-representation analysis. Results The 69% (25/36) of cells at early passages retained genetic alteration and the same DM patterns of the original tumors, with no differences related to 2D/3D methods or the presence of serum in media. The 70% (24/34) of primary cell lines analyzed at later passages (>5 or >14 days in culture) diverged from the primary tumor, the totality of those cultured with serum. All divergent cells clustered together acquiring common deregulated epigenetic signature induced by serum culture media, 2D methods and longer time in culture.
Conclusions
We have shown that global DM profiles, along with CNV analysis are useful tools to detect the recapitulation of pBT-derived primary cell-lines from the original tumor. Whatever subgroups tested, our results suggest that in vitro models should be passaged as little as possible to retain the epigenetic and genetic alterations of the tumors and thus to be considered relevant for basic and translational biology.
Abstract
BACKGROUND
In vitro models of pediatric brain tumors (pBT) are instrumental for both understanding the oncogenic molecular mechanisms and identifying/testing new therapeutic strategies. DNA ...methylation (DM) is a stable epigenetic modification recently used to classify tumors. We aim to apply DM and Copy Number Variation (CNV) profiling to characterize pBT primary cell lines and tumors.
METHODS
We included 36 pBT tissues from different histology (13 LGG, 9 DIPG, 9 HGG, 3 MB, and 2 Ependymomas), paired to their derived primary cultures. Cultures were established in two-dimensional (2D) or three-dimensional (3D) condition, as stem-cell or in serum-supplemented medium. For 9 cultures, both early (P2-P3) and long-term passages (>P4) were considered. Samples were analyzed for DM and CNV profiles using Illumina EPIC arrays and data compared with those of the brain tumor classifier.
RESULTS
At early passages all cells retained the same DM and genetic patterns of original tumors, with no differences related to 2D/3D methods or presence of serum in media. Primary cell lines analyzed at > P4 and cultured in serum diverged from the primary tumor.
CONCLUSIONS
DM profiles and CNV are useful tools to detect the recapitulation of pBT-derived primary cell-lines from the original tumor. Whatever subgroups tested, results suggest that in vitro models should be passaged as little as possible to retain the epigenetic and genetic alterations of the tumors and thus to be considered relevant for basic and translational biology. Ongoing experiments are aimed to determine how stable DM and CNV are in other conditions/tumor subgroups.
The prevalence of diabetes mellitus is increasing world-wide, even if it varies markedly in the geographical areas and populations investigated. This study is part of the Progetto Finalizzato ...Invecchiamento (Aging Project) of the Italian NCR (National Research Council) and is aimed at investigating the prevalence of diabetes and selected clinical characteristics in a study sample aged between 65 and 84 years of age resident in Catania (Italy). The prevalence rate for type II diabetes was 22.8% and it is certainly among the highest values recorded to date in other areas of Italy and abroad. We distinguished between two forms of diabetes in subjects >70 years of age: aged diabetes with onset in middle age (AD); and diabetes of senescence with onset after 70 years of age (DS). Prevalence rate was 18% for AD and 4.8% for DS, respectively. The age-specific rates of AD and DS show the progressive lower prevalence rates of the former and the higher rates of the latter. We assume that DS is mainly caused by atherosclerotic processes and represents the typical form of diabetes in the elderly.
The altered laboratory thyroid parameters indicating hypothyroidism were evaluated in a series of 3015 subjects over 65 years of age by determining total T3, total T4 and TSH. In addition, clinical ...signs of hypothyroidism were recorded in a subsample of 300 randomly selected elderly. Our results showed a high prevalence of altered laboratory thyroid parameters indicating hypothyroidism of 17.88%, whereas the real prevalence (both clinical and laboratory) is 1.00% with a female/male ratio of 2.00. The most frequent laboratory alterations was the so-called ‘alerted pituitary’ status'. The most common clinical signs of hypothyroidism involved the nervous system. We conclude that it is very difficult to diagnose hypothyroidism in the elderly and that the most indicative laboratory alterations seem to be TSH values above 3 IU/ml determined using the IRMA method.
The most common laboratory alterations of thyroid function indicating hyperthyroidism were evaluated in a series of 3015 subjects over 65 years of age by determining total T3, total T4 and TSH. ...Ultrasound and scintigraphy of the thyroid were performed where necessary. Our results showed a high prevalence of laboratory alterations hyperthyroidism type of around 8.09%, whereas the real prevalence (both clinical and laboratory) is 2.00% with a female/male ratio of 2.00. The most frequent hyperfunctioning thyreopaty is thyroidal adenoma (50%), followed by toxic multinodular goiter (33%) and Basedow's disease (17%). The most common clinical signs of hyperthyroidism involve the neuromuscular system and are often present in the so called ‘euthyroid’ elderly subject. We conclude that it is very difficult to diagnose hyperthyroidism in the elderly on clinical and laboratory grounds. The most significative laboratory thyroid parameter indicating hyperthyroidism seems to be TSH values below 0.2 IU/ml determined using the ultrasensitive IRMA method.
Senile diabetes and bone mineral density Maugeri, Domenico; Panebianco, Piera; Destro, Gera ...
Archives of gerontology and geriatrics,
05/1995, Volume:
20, Issue:
3
Journal Article
Peer reviewed
Bone mineral density was determined in a series of 67 elderly diabetics (38 males and 29 females) and 40 non-diabetic elderly subjects (20 males and 20 females) at the third medial and tenth ...ultradistal of the non-dominating radius using an X-ray densitometer (DEXA). Bone metabolism markers (Ct, PTH, HOP, UCA, AP, Vit-25-OH-D, BGP) were also measured. Our results indicate that there is no significant difference in values of BMD and the bone metabolism markers studied between diabetic and non-diabetic elderly subjects. We believe that senile diabetes is not a risk factor of onset and maintenance of senile osteoporosis.
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