MiR overexpression was shown to enhance radiosensitivity and alter target gene expression, including upregulation of PTEN, a tumour suppressor that regulates PI3K signalling. Methods: The impact of transient miR expression on radiation response was evaluated in OAC cell lines via clonogenic assay. Pharmacological inhibition of PI3K signalling using a clinically-relevant inhibitor, GDC, was evaluated as a single agent in vitro and in vivo and in combination with RT in vitro.
MiR overexpression significantly enhanced radiation sensitivity and increased PTEN expression in vitro. Treatment with GDC as a single agent led to decreased cellular viability in vitro and long-term survival in vivo. Importantly, combination treatment of GDC and radiation led to enhancement of radiosensitivity in OAC cell lines. This suggests that miR replacement therapy prior to neo-CRT, or pharmacological manipulation of downstream pathways, such as PI3K, could potentially enhance treatment efficacy.
Therefore, combination of PI3K inhibition with irradiation could be potentially explored as an additional strategy to tackle radiation resistance in non-responder OAC patients. Joshua Dulong. PhD Student. Individual radiosensitivity: establishment of a genomic signature of patients suffering from severe side effects of radiotherapy AM - PM Abstract Text Radiation therapy is a major and efficient treatment of cancer.
Biological markers are thus needed to set up new predictive tests able to identify patients at risk for normal tissue overreaction. The aim of the present study was to identify a molecular signature of clinical radiosensitivity based on new molecular mechanisms of cell sensitivity.
Skin biopsies were obtained from non-irradiated skin of these patients and dermal fibroblast cultures were established. We selected a sub-population comprising cells from 16 women treated for breast cancer, comprising grade 2 8 and grade 3 patients 8 , that we compared to cells from 8 healthy donors. Classification of global transcriptome and methylome data allowed us to clearly distinguish COP patients from the normal controls, but did not classify the patients according to their clinical grade.
We characterized a set of genes as potential markers to identify radiosensitive patients.
Moreover, all dermal fibroblasts from COP patients were found more radiosensitive than healthy donors. Our work reveals a molecular signature of radiosensitive cells from patients who suffered severe side effects of radiotherapy and paves the way to further understand the mechanisms of individual radiosensivity. However very little is known about the role of these complexes in the DNA damage tolerance response during DNA replication. Therefore, these early observations suggested a defective response to stalled replication forks in mutants that lack INO In fact, in the yeast S.
Finally, INO80 does not seem to discriminate between early and late ARS, a characteristic shared by many factors involved in the S phase checkpoint. Therefore, since HU induces the checkpoint response, and many checkpoint factors are recruited only to early ARS, INO80 binding distribution to ARS does not correspond with a checkpoint-related function, as it will be explain in the next section.
Checkpoint activation induces cell cycle delay, fork stabilization, and prevention of late firing origins activation.
Molecular determinants of post-mastectomy breast cancer recurrence
Thus, in the absence of Rad53 early replication forks collapse and late firing origins repression is lost Branzei and Foiani Interestingly, the involvement of INO80 in the S phase checkpoint seems to be controversial, since analysis of Rad53 phosphorylation in the ino80 mutant by different research groups has shown dissimilar results.
Moreover, this group also described that Rad53 inactivation is delayed in the 94 K. Therefore, available evidence indicates that INO80 does not seem to affect replication fork stabilization after HU treatment. As such, the hypersensitivity of the ino80 mutant to HU on plates, which is not connected to the defect in replication fork stabilization shown by 2D gel electrophoresis, is known to be a characteristic of mutants of genes that mediate DNA damage tolerance during replication.
Therefore, as it will be discussed in the next section, INO80 has a pivotal role in the DNA damage tolerance pathways during replication. Thus, if the obstruction is in the template for the leading strand, a gap is formed, and the subsequent excision of this region creates a DSB by destruction of the replication fork leading to H2AX phosphorylation.
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Furthermore, analysis of S phase synchronized cells treated with MMS by two different techniques confirms these results. Therefore, the altered chromosome mobility of arp8 cells in PFGE after MMS treatment is due to the persistence of unreplicated gaps that represents an impaired ability of the mutants to complete DNA replication.
In fact, pull down from trichloroacetic acid extracted proteins followed by western blot analysis using an anti-Ub antibody in the ino80 mutant, shows that in S phase synchronized MMS-treated cells, the ubiquitylated forms of PCNA are significantly lower in abundance as compared to the wild-type strain. Rad51 activity at forks generates DNA hemicatenate-like structures called X-shaped structures.
Thus, it is possible that INO80 binds to replication forks, and remodels the chromatin to allow proper recruitment of DNA damage tolerance factors like Rad18 and Rad Moreover, Rad51 recruitment to ARS is, in fact, significantly reduced in the ino80 mutant, when assessed by chromatin immunoprecipitation analysis. Rad51 recruitment leads to activation of recombinationmediated processes that possibly, as part of a template switching mechanism, will lead to fork resolution and DNA replication completion without the generation of DSBs, genomic aberrations, and genomic instability Fig.
When replication forks encounter obstructions caused by DNA damage the INO80 chromatin remodeling complex is recruited to blocked replication forks. From epigenetic modifications to nucleosome mobilization, it is now clear that chromatin structure and its respective modifiers are essential players in a multitude of DNA-related activities. Specifically, chromatin remodeling complexes, through their ability to remodel the structure of chromatin, act as DNA gatekeepers, granting or denying access to the molecule and, thus, regulating many nuclear functions. In particular, one remodeling complex, INO80, has been implicated in several unrelated novel nuclear activities, constituting a good example of the extreme versatility these complexes have achieved through evolution.
Yet, it is known that each different subunit has its own specific function, but it is also clear that they work together to perform different tasks.
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In addition, the presence of posttranslational modifications in some subunits adds a different level of complexity to the picture, related to the possible regulatory mechanisms that could be directing INO80 activities. Clearly, further research would certainly advance our knowledge of this complex as well as the intricate relations established among its subunits.
Therefore, further exploration of this new research area could bring innovative and invaluable tools to improve the knowledge and treatment of diseases such as cancer, where accurate DNA replication and proper activation of DNA damage avoidance pathways have an important impact on the disease progression. Mutat Res —29 Branzei D, Foiani M Interplay of replication checkpoints and repair proteins at stalled replication forks.
Nature — Lundblad V, Blackburn EH An alternative pathway for yeast telomere maintenance rescues est1- senescence. Cell — Lundblad V, Szostak JW A mutant with a defect in telomere elongation leads to senescence in yeast. Science — Takata H, Tanaka Y, Matsuura A Late S phase-specific recruitment of Mre11 complex triggers hierarchical assembly of telomere replication proteins in Saccharomyces cerevisiae. Mol Cell Biol — K.
Cooperation between histone chaperones and ATPdependent nucleosome remodeling machines. Here we provide a comprehensive review of C. To better explain C.
Parallel session 5: Genomic determinants: Radiation response and toxicity
The ionizing radiation IR response is no exception, and early studies focused on the effect of DNA damage on single cell organisms such as yeasts. Using the budding yeast Saccharomyces cerevisiae as a model system has been a powerful method to identify genes required for DNA damage repair. Most of the key factors in this process have been isolated by performing forward genetic screens for mutants sensitive to DNA-damaging agents Weinert and Hartwell However, yeast cells lack an important aspect of the DNA damage response present in higher eukaryotic organisms — the removal of genetically compromised cells by programmed cell A.
Bailly and A. Gartner death apoptosis.
It is still technically challenging to perform forward genetic screens with vertebrate cell lines, and large-scale RNAi screening procedures do not permit the generation of stable mutations or hypomorphic alleles in essential genes. Therefore, the use of a model system that recapitulates the integral DNA damage response of mammalian cells but concurrently provides the ease of maintenance and manipulation of a small and simple organism is of great interest to the scientific community.
Over the past years an increasing number of laboratories have engaged in establishing and exploiting the nematode worm Caenorhabditis elegans as an experimental system to study DNA damage response genes. Before going into the details of C.
A basic knowledge of worm physiology and life cycle is also necessary to comprehend the various strategies used by somatic and germ cell tissues to respond to DNA damage, as well as to understand the basic assays that are used to assess defects in DNA damage response pathways. Studies mostly focus on worm development and are aided by the invariant somatic cell lineage. All individual animals display the same cell division patterns to produce identical groups of differentiated cells, and the complete process of embryogenesis can be monitored by light microscopy.
The pioneering work of Sulston and co-workers established the complete map of the C. Knowledge of the invariant lineage enables using the worm to ask basic biological questions, such as the molecular details of inductive developmental processes at the level of individual cells.
During larval development, further cell divisions occur in somatic tissues, especially in the ventral cord and during development of the vulva. However, most cell proliferation happens in the C. In the adult all somatic cells are post-mitotic and the germline is the only proliferative tissue. Schematic representation of a C. The distal end of the worm germline is comprised of mitotic cells.
Nuclei arrow and the surrounding cytoplasm are enlarged in irradiated cells due to continued cellular growth in the absence of cell division A. Gartner considering radiation responses in adult worms. It is thus not surprising that somatic tissues tolerate higher levels of DNA damage and use other DNA repair strategies compared to germ cells. Germlinespecific checkpoints and accurate recombinational repair are used to guard germ cells from acquiring deleterious mutations that could be passed on to the next generation.
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Germ cell nuclei are actually not fully cellularized and are thus part of a syncytium, although for convenience they are referred to as cells both in this review and generally in the C. Proximal to the transition zone most cells are in meiotic pachytene; homologous chromosomes are tightly aligned to each other forming the synaptonemal complex. Germ cells subsequently complete meiosis and concomitantly undergo cellularization. Worms achieve this by temporally switching the sex of the germ line Hodgkin and Brenner The germline acquires a male fate during the L4 larval stage and all germ cells differentiate into amoeboid sperm cells, stored in an organ termed the spermatheca.
Oocytes are produced when the germline switches to its female fate towards the end of the L4 larval stage. In adult worms, fertilization occurs when oocytes pass through the spermatheca, allowing oogenesis to occur at the proximal end of the gonad Fig. Failure to undergo meiotic recombination does not lead to overt changes in germ cell identity or morphology, and meiotic chromosome pairing is not affected. Mutants defective in recombinational repair of DNA DSBs often also show defects in meiotic recombination, leading to defects in meiotic crossover formation and meiotic chromosome segregation, and resulting in embryonic lethality in the following generation.