scholarly journals Διερεύνηση μηχανισμών χρωμοσωμικής αστάθειας και τελομερικής ομοιόστασης κατά την επαγωγή καρκινικών βλαστικών κυττάρων in vitro και in vivo

2019 ◽  
Author(s):  
Φανή-Μάρλεν Ρουμελιώτη
Keyword(s):  
Chromosomal Instability ◽  
Alternative Lengthening Of Telomeres ◽  
Alternative Lengthening

Ως χρωμοσωμική αστάθεια στη νεοπλασία (CIN-Chromosomal Instability in Neoplasia) ορίζεται ο αυξημένος ρυθμός συνεχών απωλειών ή διπλασιασμών ολόκληρων χρωμοσωμάτων ή μεγάλων τμημάτων τους κατά την κακοήθη κυτταρική ανάπτυξη. Η χρωμοσωμική αστάθεια είναι πολύ συχνή στη νεοπλασία (παρατηρείται περίπου στο 90% των συμπαγών όγκων και σε πολλές αιματολογικές κακοήθειες). Η CIN είναι συνεχής διαδικασία, διότι κατά την εξέλιξη της νόσου, ενισχύεται διαρκώς από την έκθεση των πολλαπλασιαζόμενων νεοπλασματικών κυττάρων σε τελομερική δυσλειτουργία, στρες αντιγραφής, ανεπάρκεια των σημείων ελέγχου του κυτταρικού κύκλου κύκλου και διαταραχές της μιτωτικής ατράκτου. Η τελομερική δυσλειτουργία και πως αυτή επηρεάζει τη χρωμοσωμική αστάθεια, είναι το κύριο θέμα έρευνας της παρούσας διατριβής. Τα τελομερή είναι εξειδικευμένες δομές που παίζουν σημαντικό ρόλο στη σταθερότητα του χρωμοσώματος. Η διασφάλιση της λειτουργικής και δομικής ακεραιότητάς τους έχει ζωτική σημασία για τη φυσιολογική και παθολογική κυτταρική λειτουργία. Η ικανότητα συνεχούς κυτταρικού πολλαπλασιασμού στον καρκίνο είναι άμεσα εξαρτώμενη από την ενεργοποίηση των μηχανισμών αναπλήρωσης των τελομερών. Στις νεοπλασίες των θηλαστικών, το μήκος των τελομερών ανανεώνεται κυρίως από το ένζυμο τελομεράση (TERT) ή σπανιότερα μέσω της εναλλακτικής επιμήκυνσης των τελομερών (alternative lengthening of telomeres-ALT). Στην ανθρώπινη νεοπλασία, ο συνεχής κυτταρικός πολλαπλασιασμός απουσία τελομεράσης (ALT) χαρακτηρίζεται από υψηλούς ρυθμούς δομικής αστάθειας των χρωμοσωμάτων. Αυτό οφείλεται στο ότι τα χρωμοσώματα των ALT καρκινικών κυττάρων δε διαθέτουν επαρκή τελομερική προστασία και είναι συχνά επιδεκτικά σε τυχαίες τελομερικές συντήξεις. Οι συντήξεις των τελομερών οδηγούν σε φαινόμενα γενικευμένης γενωμικής αστάθειας και οδηγούν σε ανασυνδυασμούς μεταξύ χρωμοσωμάτων οι οποίες ενεργοποιούν ή καταστέλλουν αντίστοιχα, ογκογόνα ή ογκοκατασταλτικά γονίδια. Τα νεοπλασματικά κύτταρα που χρησιμοποιούν τον ALT μηχανισμό επιμήκυνσης των τελομερών παρουσιάζουν 5 φορές αυξημένη συχνότητα χρωμοσωμικής αστάθειας σε σχέση με αυτά που χρησιμοποιούν την τελομεράση.Σκοπός της παρούσας διδακτορικής διατριβής είναι η διερεύνηση μηχανισμών δημιουργίας ανευπλοειδίας και πολυπλοειδίας κατά την εξέλιξη ALT καρκινικών κυττάρων σε συνθήκες γενοτοξικού στρες και στρες της αντιγραφής, σε μια προσπάθεια συμβολής στην κατανόηση της χρωμοσωμικής αστάθειας προς την ανάπτυξη νέων, αποτελεσματικότερων προγνωστικών διαγνωστικών ή ογκοθεραπευτικών μεθόδων.

scholarly journals Alternative lengthening of telomeres is associated with chromosomal instability in osteosarcomas

Oncogene ◽  
2001 ◽  
Vol 20 (29) ◽  
pp. 3835-3844 ◽  
Author(s):  
Christina Scheel ◽  
Karl-Ludwig Schaefer ◽  
Anna Jauch ◽  
Monika Keller ◽  
Daniel Wai ◽  
...  
Keyword(s):  
Chromosomal Instability ◽  
Alternative Lengthening Of Telomeres ◽  
Alternative Lengthening

scholarly journals ATRX alteration contributes to tumor growth and immune escape in pleomorphic sarcomas

2020 ◽  
Author(s):  
Lucie Darmusey ◽  
Gaëlle Pérot ◽  
Noémie Thébault ◽  
Sophie Le Guellec ◽  
Nelly Desplat ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Immune Escape ◽  
Adaptive Immune System ◽  
Tumor Growth Rate ◽  
Adaptive Immune ◽  
Expression Of Genes ◽  
Alternative Lengthening ◽  
Poorly Differentiated

AbstractWhole genome and transcriptome sequencing of a cohort of 67 leiomyosarcomas revealed ATRX to be one of the most frequently mutated genes in leiomyosarcomas after TP53 and RB1. While its function is well described in the alternative lengthening of telomeres mechanism, we wondered whether its alteration could have complementary effects on sarcoma oncogenesis. ATRX alteration is associated with the down-expression of genes linked to differentiation in leiomyosarcomas, and to immunity in an additional cohort of 60 poorly differentiated sarcomas. In vitro and in vivo models showed that ATRX loss increases tumor growth rate and immune escape by decreasing the immunity load of active mast cells in sarcoma tumors. These data indicate that an alternative to unsuccessful targeting of the adaptive immune system in sarcoma could be to target the innate system. This might lead to a better outcome for sarcoma patients in terms of ATRX status.

Integrating CRISPR screening with tumor genomic analyses to identify therapeutic targets in hepatocellular carcinomas (HCC) independent of P53.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e14659-e14659
Author(s):  
Ankur Sheel ◽  
SuetYan Kwan ◽  
Wen Xue
Keyword(s):  
Cell Proliferation ◽  
Cell Growth ◽  
Cell Lines ◽  
Chromosomal Instability ◽  
Cancer Genomics ◽  
Essential Gene ◽  
Patient Data ◽  
Underlying Mechanisms

e14659 Background: Hepatocellular carcinoma (HCC) is an aggressive subtype of liver cancer with few effective treatments. Moreover, the underlying mechanisms that drive HCC pathogenesis remain poorly characterized. Identifying genes and pathways essential for HCC cell growth will aid the development of new targeted therapies for HCC. Furthermore, the P53 pathway is frequently mutated in HCC therefore identifying targets with therapeutic efficacy irrespective of P53 status is warranted. Methods: To identify kinases essential for HCC proliferation, we performed a kinome wide CRISPR screen in human HCC cell lines with varying P53 mutations and validated our findings using CRISPR-Cas9 mediated genetic manipulations in human HCC cell lines in-vitro and in-vivo. Furthermore, we performed an integrated cancer genomics analyses using patient data from TCGA and the NCI to validate the relevancy of our findings. Results: We identified transformation/transcription domain-associated protein (TRRAP) as an essential gene for HCC cell proliferation. we show that depletion of TRRAP or its co-factor, histone acetyltransferase KAT5, inhibits HCC cell growth via induction of P53, P21 and RB-independent senescence in-vitro and in-vivo. Furthermore, we find that TRRAP is upregulated in HCC patient samples independent of TP53 mutations. Integrated cancer genomics analyses using both HCC patient data derived from TCGA and from RNA-sequencing of our in-vitro model identified a chromosomal instability signature that was regulated by TRRAP/KAT5 in-vitro. Furthermore this chromosomal instability signature was also upregulated in HCC patients. Finally, we identify TOP2A as a target in this pathway as genetic depletion of TOP2A inhibited cell growth via induction of senescence. Conclusions: Our results uncover a role for TRRAP/KAT5 in promoting HCC cell proliferation via activation of mitotic genes in order to potentiate a chromosomal instability signature. Our findings suggest that targeting the TRRAP/KAT5 complex and TOP2A is a therapeutic strategy for HCC, even in tumors that have escaped P53 and RB tumor suppressive programs.

Sumoylation of TRF1 Is Essential for Its Recruitment to ALT-Associated PML Bodies.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4169-4169
Author(s):  
Jian Yu ◽  
Jianping Lan ◽  
Yuanyuan Zhu ◽  
Xiaoyu Lai ◽  
He Huang
Keyword(s):  
Cancer Cells ◽  
Computational Prediction ◽  
Proliferative Potential ◽  
Telomeric Dna ◽  
Alternative Lengthening ◽  
Pml Bodies ◽  
U2os Cells ◽  
Telomeric Binding Protein

Abstract To achieve unlimited proliferative potential, most cancer cells activate telomerase to maintain telomeres. However, some cancer cells elongate telomeres through a telomerase-independent pathway termed alternative lengthening of telomeres (ALT). These ALT cells contain a novel promyelocytic leukemia (PML) body (ALT-associated PML body, APB), which comprises telomeric DNA and a number of proteins, including PML protein, the telomere binding proteins TRF1 and TRF2, replication factor A, and recombination factors Rad51, Rad52, and the Rad50/Mre11/NBS1 complex. TRF1, as the first identified telomeric binding protein, binds the duplex telomeric repeats at telomere ends. It plays an important role in telomere length control, telomeric ends shelter and cell cycle regulation. In ALT cell lines, TRF1 co-localized with PML protein at APBs, but the exact mechanism of its recruitment to APBs is not clear. Here we show that TRF1 localizes to PML bodies in about 5% of an asynchronously growing culture of U2OS cells and the percentage of cells with colocalization of TRF1 and PML bodies increases to about 40% in cells enriched in G2/M, which is consistent with the previous studies. Furthermore, our results show that TRF1 is modified by the small ubiquitin-like protein SUMO-1 in vivo and in vitro. Firstly, 293T cells were transfected with Flag-TRF1, HA-Ubc9, GFP-SUMO1 and then immunoprecipitated by using FLAG-M2 gel under denaturing conditions. Immunoblotting with GFP and Flag antibodies demonstrated that TRF1 is modified by SUMO-1 in vivo. Next, in vitro SUMO-1 conjugation assay of TRF1 was employed. The results showed that TRF1 is conjugated with SUMO-1 in the presence of purified recombinant protein SAE1/SAE2, Ubc9, SUMO1, His-TRF1 and ATP. Either SAE1/SAE2, Ubc9, SUMO-1 or ATP was omitted from the reaction abolished the sumoylation of TRF1. Previous studies have shown that sumoylation controls the recruitment of several proteins to PML bodies, so we examined whether sumoylation of TRF1 is required for its recruitment to APBs. We mutated the potential sumoylation sites of TRF1 according to the computational prediction and then transfected it into U2OS cells to examine its localization. The results showed that TRF1 mutant does not localize to PML bodies. Taken together, all these results suggest that TRF is modified by SUMO-1 and sumoylation of TRF1 is essential for its recruitment to APBs in ALT cells.

scholarly journals Hypo-methylation mediates chromosomal instability in pancreatic NET

2017 ◽  
Vol 24 (3) ◽  
pp. 137-146 ◽  
Author(s):  
I Marinoni ◽  
A Wiederkeher ◽  
T Wiedmer ◽  
S Pantasis ◽  
A Di Domenico ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Chromosomal Instability ◽  
Global Dna Methylation ◽  
Epigenetic Changes ◽  
Knock Down ◽  
Alternative Lengthening ◽  
Increased Risk ◽  
Pancreatic Net ◽  
G0 Phase

DAXX and or ATRX loss occur in 40% of pancreatic neuroendocrine tumors (PanNETs). PanNETs negative for DAXX or ATRX show an increased risk of relapse. The tumor-associated pathways activated upon DAXX or ATRX loss and how this event may induce chromosomal instability (CIN) and alternative lengthening telomeres (ALT) are still unknown. Both DAXX and ATRX are involved in DNA methylation regulation. DNA methylation of heterochromatin and of non-coding sequences is extremely important for the maintenance of genomic stability. We analyzed the association of DAXX and/or ATRX loss and CIN with global DNA methylation in human PanNET samples and the effect of DAXX knock-down on methylation and cell proliferation. We assessedLINE1as well as global DNA methylation in 167 PanNETs, and we found that DAXX and or ATRX-negative tumors and tumors with CIN were hypomethylated. DAXX knock-down in PanNET cell lines blocked cells in G1/G0 phase and seemed to increase CIN in QGP-1 cells. However, no direct changes in DNA methylation were observed after DAXX knock-downin vitro. In conclusion, our data indicate that epigenetic changes are crucial steps in the progression of PanNETs loss and suggest that DNA methylation is the mechanism via which CIN is induced, allowing clonal expansion and selection.

scholarly journals Involvement of Poly(ADP-Ribose) Polymerase 1 and Poly(ADP-Ribosyl)ation in Regulation of Centrosome Function

2003 ◽  
Vol 23 (7) ◽  
pp. 2451-2462 ◽  
Author(s):  
Masayuki Kanai ◽  
Wei-Min Tong ◽  
Eiji Sugihara ◽  
Zhao-Qi Wang ◽  
Kenji Fukasawa ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Chromosomal Instability ◽  
Regulatory Mechanism ◽  
Parp Inhibitor ◽  
Daughter Cells ◽  
P53 Tumor Suppressor Protein ◽  
Centrosomal Proteins ◽  
Parp 1

ABSTRACT The regulatory mechanism of centrosome function is crucial to the accurate transmission of chromosomes to the daughter cells in mitosis. Recent findings on the posttranslational modifications of many centrosomal proteins led us to speculate that these modifications might be involved in centrosome behavior. Poly(ADP-ribose) polymerase 1 (PARP-1) catalyzes poly(ADP-ribosyl)ation to various proteins. We show here that PARP-1 localizes to centrosomes and catalyzes poly(ADP-ribosyl)ation of centrosomal proteins. Moreover, centrosome hyperamplification is frequently observed with PARP inhibitor, as well as in PARP-1-null cells. Thus, it is possible that chromosomal instability known in PARP-1-null cells can be attributed to the centrosomal dysfunction. P53 tumor suppressor protein has been also shown to be localized at centrosomes and to be involved in the regulation of centrosome duplication and monitoring of the chromosomal stability. We found that centrosomal p53 is poly(ADP-ribosyl)ated in vivo and centrosomal PARP-1 directly catalyzes poly(ADP-ribosyl)ation of p53 in vitro. These results indicate that PARP-1 and PARP-1-mediated poly(ADP-ribosyl)ation of centrosomal proteins are involved in the regulation of centrosome function.

scholarly journals CBMT-19. THE ALTERNATIVE LENGTHENING OF TELOMERE (ALT) MECHANISM PROVIDES COLLATERAL SENSITIVITY TO LETHAL TELOMERIC FUSION INDUCED BY TRAPPING PARP INHIBITORS

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi37-vi37
Author(s):  
Joydeep Mukherjee ◽  
Cecelia Dalle-Ore ◽  
Tor-Christian Johanessen ◽  
Ajay Pandita ◽  
Shigeo Ohba ◽  
...  
Keyword(s):  
Dna Damage ◽  
Genomic Dna ◽  
Parp Inhibitors ◽  
Parp Inhibition ◽  
Collateral Sensitivity ◽  
Astrocytoma Cells ◽  
Alternative Lengthening ◽  
New Treatment

Abstract A subset of human tumors, including all IDH1-mutant astrocytomas, use a homologous recombination-based alternative lengthening of telomere (ALT) pathway to resolve telomeric dysfunction in the absence of TERT. Because ALT is not used by normal cells, targeting of the process may provide new therapeutic options for patients with ALT-dependent tumors. We here report that reliance on the ALT mechanism makes tumors collaterally hypersensitive to clinically-available trapping PARPi (t-PARPi). Specifically we noted that astrocytoma cells dependent on the ALT-mechanism (IDH1-mutant and ATRX-deficient genetically-modified human astrocytes and MGG119 PDX) were significantly more sensitive to trapping PARPi than matched ALT-independent isogenic ATRXWT astrocytes and MGG152 PDX cells, respectively) both in vitro and in vivo. Surprisingly this hypersensitivity was not associated with BRCA-ness, the extent of PARP inhibition, or with t-PARPi-created genomic DNA damage as is the case in most PARPi-sensitive populations. Rather the enhanced activity of t-PARPi in ALT-dependent cells was associated with a novel t-PARPi-induced, lethal telomere fusion. Furthermore, the extent of chromosomal fusion was proportional to the PARP-trapping ability of the five PARP inhibitors tested, and could be prevented by exogenous expression of TERT, which eliminated reliance on ALT but did not alter levels of PARPi-induced genomic DNA damage. The extent of tPARPi-induced telomeric fusion in ALT-dependent cells, which could be directly measured in small amounts of DNA using a q-PCR approach, was also directly proportional to tPARPi-induced cell death in vitro and to prolonged survival of tumor-bearing mice in vivo. These results therefore identify clinically available tPARPi as a new treatment modality for a select and easily genetically definable group of ALT tumors, and also define telomeric fusion as a biomarker of drug action in these tumors.

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