359 research outputs found
In vitro chromosomal radiosensitivity in breast cancer patients
No full textIn vitro chromosomal radiosensitivity has been investigated in patients with inherited cancer prone syndromes (e.g. Ataxia Telangiectasia, Nijmegen breakage syndrome) and also in different types of cancer patients (e.g. head and neck-, colorectal cancer, breast cancer). In breast cancer patients enhanced chromosomal radiosensitivity is observed in several independent studies. In most of these studies chromosomal radiosensitivity was analysed by using the G2 and G0-MN assay for peripheral blood lymphocytes and it has been suggested that enhanced chromosomal radiosensitivity of PBL may be a marker for breast cancer predisposing genes of low penetrance. In the first part of this thesis we investigated the suitability of the G2 and G0-MN assay as biomarkers of individual radiosensitivity. Therefore we analysed the reproducibility and sensitivity of these assays. Our studies showed that the intra-individual variability of both assays is high and not different from the inter-individual variability. Although it was suggested in literature that the inter-individual variability might be attributable to the hormonal status in females, we could not demonstrate any influence of the blood serum levels of estradiol or progesterone on the intra-individual and inter-individual variability obtained in the G0-MN assay. In most of the studies investigating the in vitro chromosomal radiosensitivity of breast cancer patients the G0-MN assay has been performed on fresh blood cultures. Because the use of fresh blood cultures allows no repeated testing, we have investigated the suitability of EBVtransformed cell lines and IL-2 cultures for the analysis of chromosomal radiosensitivity in breast cancer patients. As we were not able to demonstrate the radiosensitive response in the cell cultures derived from the original PBL, care has to be taken when EBV-transformed cell lines or IL-2 cultures are used to assess chromosomal radiosensitivity in breast cancer patients.In the second part of this thesis, we analysed chromosomal radiosensitivity of different breast cancer populations with the G2 and G0-MN assay. We have demonstrated that a group of breast cancer patients with a known or putative genetic predisposition is more radiosensitive than a group of healthy women and this as well with the G2 and G0-MN assay. We showed that this enhanced in vitro chromosomal radiosensitivity is not correlated with the age of onset of the disease in breast cancer patients and that the enhanced chromosomal radiosensitivity in breast cancer patients with a mutation in BRCA1 or BRCA2 is not due to the presence of a
mutation in these two genes. Analysis of the in vitro chromosomal radiosensitivity of a small group of breast cancer patients with a CHEK2 1100delC mutation revealed no correlation between the CHEK2 1100delC variant and chromosomal radiosensitivity. No correlation is found between the G2 and G0 chromosomal radiosensitivity, pointing to the fact that these two assays reflect different underlying mechanisms of chromosomal radiosensitivity. In conclusion, we performed a thorough analysis of the suitability of the G2 and G0-MN assay as biomarkers of individual radiosensitivity and we applied these assays to determine the chromosomal radiosensitivity of different population groups of breast cancer patients. We can conclude that both assays are very valuable to analyse chromosomal radiosensitivity in breast cancer populations. Further studies have to be performed to analyse the underlying mechanisms of chromosomal radiosensitivity
The role of the Ku heterodimer in the response of human MCF10A cells to low-and high-LET radiation qualities
No full textSeveral observations suggest that the mechanisms involved in DNA double-strand break (DSB) repair are of particular importance during breast tumorigenesis and chromosomal radiosensitivity. An enhanced chromosomal radiosensitivity has been observed in a large number of breast cancer patients by means of the G0 micronucleus (MN) and G2 chromatid break assay. As micronuclei and chromatid breaks are the result of non-repaired or misrepaired DNA DSBs, mutations and/or polymorphisms in DNA DSB repair genes could account for the observed elevation in chromosomal radiosensitivity in breast cancer patients and breast cancer risk. Recently, our research group showed that single nucleotide polymorphisms (SNPs) in the DSB repair genes, Ku70 and Ku80, are associated with an enhanced breast cancer susceptibility and in vitro chromosomal radiosensitivity. To further unravel the underlying mechanisms responsible for an enhanced chromosomal radiosensitivity in breast cancer patients and breast cancer risk, we investigated in a first section of this dissertation the role of the Ku70/80 (Ku) heterodimer, in the response of human breast epithelial cells to low-LET (linear energy transfer) 60Co γ-rays. For this purpose, we generated a stable Ku-knockdown by lentiviral-mediated RNAi of Ku70 in human breast epithelial MCF10A cells. Analysis of chromosomal radiosensitivity by means of the MN assay in both knockdown and control MCF10A cells showed that knockdown of Ku70/80 caused a significant increase in the number of micronuclei, which supports the hypothesis that defects in DNA DSB repair underlie chromosomal radiosensitivity. We further demonstrated that this increase in chromosomal radiosensitivity was accompanied by a significant decrease in cell survival, measured by both the crystal violet cell proliferation assay and the classic colony formation assay. To further understand the mechanisms that lead to this radiosensitive phenotype, DSB induction and repair was investigated using γH2AX foci as a marker of DNA DSBs. These results showed that downregulation of Ku resulted in a higher number of foci present at early post-irradiation times, which could be further indicative of less efficient repair mechanisms. A profound knowledge of the pathways involved in the cellular response to radiation-induced damage is not only important for an improved understanding of the mechanisms underlying breast cancer susceptibility, it also has important implications for the use of ionizing radiation in radiotherapy as the ability to repair radiation-induced damage will determine therapeutic outcome of both normal and malignant cells. Besides the use of conventional low-LET radiation in radiotherapy, high-LET radiation qualities are increasingly implemented. Based on the argument that depending on the radiation quality, the degree of complexity of radiationinduced DSBs differs, it could be anticipated that the relative importance of pathways involved in the repair of DNA damage also varies. Therefore, in a second section of this dissertation, we investigated the influence of Ku-knockdown in the cellular response to low-LET X-rays and high-LET neutrons, both routinely used in clinical practice. The results of this study showed that knockdown of the Ku heterodimer enhanced the chromosomal and cellular radiosensitivity of MCF10A cells to both low-LET X-rays and high-LET neutrons. Moreover, we observed that the Ku heterodimer is of similar importance for repairing radiation damage induced by both radiation qualities. These results may open perspectives towards the use of strategies to enhance radioresponsiveness of neoplastic tissues and improve the outcome of radiotherapy using both low-LET and high-LET radiation types
Image quality evaluation in X-ray medical imaging based on Thiel embalmed human cadavers
No full textPolymorphisms in DNA double-strand break repair genes: link with breast cancer susceptibility and in vitro chromosomal radiosensitivity.
No full textBreast cancer is one of the most common types of neoplasia in females in Western industrialised countries. In Belgium, breast cancer is the leading cause of death by cancer in females and the risk of being diagnosed with breast cancer before the age of 75 years in Belgium is 11.5 %. One of the strongest risk factors is a family history of the disease, indicating a genetic predisposition to breast cancer. However, only 6% of all breast cancer cases can be linked to monogenic, germline mutations in the breast cancer predisposing genes BRCA1, BRCA2, ATM, CHK2, PTEN and TP53. Epidemiological analyses suggest that the remaining breast cancer cases can be explained by a polygenic model that states that the combined effect of many individual weak genetic variants is responsible for an enhanced breast cancer risk. As enhanced in vitro chromosomal radiosensitivity is a hallmark for breast cancer and results from non‐ or misrepaired double strand breaks (DSBs), single nucleotide polymorphisms (SNPs) in DSB repair genes, could be involved in in vitro chromosomal radiosensitivity and genetic predisposition to breast cancer. Several population based case‐control studies have already shown a link between SNPs in DSB repair genes and breast cancer risk. Moreover, the breast is a selected micro‐environment, vulnerable to endogenous oxidative stress through hormone exposure. Especially oestrogen has attracted considerable attention, as it induces DSBs during its metabolism and may act as a complete carcinogen. In the studies presented in this thesis, we investigated whether SNPs in the core repair genes of DSBs are associated with an enhanced breast cancer susceptibility and/or in vitro chromosomal radiosensitivity. Genes from the two main DSB repair pathways were studied: Ku70, Ku80 and DNAPKCS of the non homologous end‐joining (NHEJ) pathway and RAD51, XRCC3, BRCA1 and BRCA2 of the homologous recombination (HR) pathway.
The results demonstrate that the c.‐1310C>G SNP in the promoter region of Ku70 is significantly associated with breast cancer risk in an unselected patient population, comprising mainly of sporadic patients. Additionally, the combination of the variant “G” allele of this polymorphism with a hormonal breast cancer risk factor, reflecting susceptibility to oestrogen exposure, is associated with a more pronounced increase in breast cancer risk. The c.2099‐2408G>A SNP in Ku80 shows a positive association with breast cancer risk in a group of patients with a known or putative genetic predisposition to the disease. Both the c.‐1310C>G (Ku70) and c.2099‐2408G>A (Ku80) point‐variations can be considered risk alleles for breast cancer and they also show a positive association with chromosomal radiosensitivity. The combination of 2 or 4 putative high‐risk genotypes in RAD51 and XRCC3 resulted in a significant association with breast cancer risk in a patient population selected for a genetic predisposition, which is in agreement with the polygenic model for breast cancer initiation. Our results are also indicative of a modifying effect of SNPs in RAD51, XRCC3, BRCA1 and BRCA2 on breast cancer penetrance and phenotype in patients carrying a pathological mutation in BRCA1 or BRCA2. We also showed that the c.190T>C variation in the BRCA1 RING finger domain may induce modifications of the protein structure which could disrupt the BRCA1‐BARD1 interaction and hence predispose to breast cancer. The studies performed in the frame of this thesis contribute to the ongoing research concerning the genetic profiles associated with an enhanced breast cancer risk. A better understanding of the underlying genetic factors, responsible for breast cancer predisposition will improve our understanding of the mechanisms underlying breast cancer aetiology and this will influence the approach to breast cancer prevention and treatment
The cellular response to different radiation qualities in relation to breast cancer screening and treatment
No full textBiomarker investigation of the health effects of CT X-ray exposure in children : a plea to 'image gently'
No full textBiological responses after low doses X-ray exposure: gene expression and mechanistic studies
No full textThe health effects arising from exposure to low doses of ionizing radiation are increasing due to the extensive use of medical and diagnostic applications. One of the central questions in the field of radiation biology is understanding the health consequences of these low doses, where several factors like genetics, life-style, and lack of robust epidemiological studies are impeding well-validated conclusions. Within the context of this PhD, we aim to understand the biological responses after low dose exposures in both in vitro and in vivo studies.
The first part of this PhD is composed of five different chapters that aim to introduce the main objectives of our research and discuss the complexity of low dose research.
Ionizing radiation contributes to the development in many of the sectors like medical diagnosis, therapy, industry, and agriculture. However, exposure to these radiations can carry serious health consequences, like cancer or non-cancer diseases. In Chapter 1, we give a short overview about the physical properties of ionizing radiation and basic concepts of dosimetry. Furthermore, we discuss the main sources of ionizing radiation and the epidemiological health studies that aimed to investigate the effects of ionizing radiation. Moreover, we discuss both deterministic and stochastic effects. On the other hand, we give a thorough overview about the risk assessment models used to estimate the risk after ionizing radiation exposures. In particular, we explain the linear-no threshold (LNT) hypothesis, which is based on linear extrapolation from high to low doses; however, several controversies challenge this hypothesis, mainly due to mechanisms involved in cellular communications involved in the low dose range.
DNA double strand breaks may induce mutations and contribute to the development of cancers if misrepaired or unrepaired. In Chapter 2, we give a general overview about the cellular and molecular mechanisms involved in radiation responses. These include DNA damage signalling and repair. Furthermore, we discuss the different mechanisms involved in radiation-induced cell death, such as apoptosis and senescence.
One of the challenging objectives within the context of low dose studies is the characterization of low dose specific biomarkers that are sensitive and robust. In Chapter 3, we highlight the basic considerations for choosing a biomarker for epidemiological studies. On the other hand, we discuss possible biomarkers that can be of use in the low dose range. DNA double strand break studies, via scoring of the Ser 139 phosphorylated form of the histone H2AX (γ-H2AX) is one of the most sensitive biomarkers for DNA damage and has been shown to be sensitive for low doses; but also, oxidative stress biomarkers must be investigated as well. Along with DNA damage studies, transcriptional and translational changes could provide "rich" information about the mechanisms involved in low dose responses, in particular whole genome analysis and cytokine measurement were shown to be reliable and sensitive techniques for radiation low dose research.
Our research has shown that immune responses play a central role after low dose exposures. In Chapter 4, we give an overview about the different immune modulatory responses involved in radiation responses. First, we discuss the radiosensitivity of the different immune blood cells followed by an overview about the basic immune pathways, such as NF-κB, MAPK and toll-like receptor (TLR) signalling and their involvement in radiation responses.
The introduction part is complemented with Chapter 5 that discusses the aims of the thesis and the outline of the research.
The second part of this PhD is composed of three different scientific papers that discuss the results combined for this dissertation.
To understand better the differences in responses between low and high doses of ionizing radiation, aliquots of whole blood samples were collected from ten healthy donors and irradiated in vitro with high and low doses of ionizing radiation. Using whole genome analysis and different bioinformatics approaches, we unravelled that low doses of ionizing radiation are characterized by the induction of an immune response by the activation of chemokine and cytokine signalling; furthermore, there they exhibit a pro-inflammatory response due to the activation of NF-κB, MAPK and TLR signalling. On other hand, the high dose response is characterized by damage signalling, mainly by the involvement of p53. Several genes belonging to immune and damaging responses were confirmed with quantitative RT-PCR. The results of this study are discussed in Chapter 6.
In an aim to confirm our in vitro whole genome analysis, we investigated the response to low doses of ionizing radiation in vivo. Because samples collected from human would be more reliable than animal studies, we chose prostate cancer patients undergoing intensity modulated radiotherapy (IMRT) as target population. These patients are adequate for low dose studies because they receive a high dose to the tumour; however, large volumes of normal tissues receive low doses. In an attempt to unravel the biological responses, whole blood samples were collected before and after the first fraction of IMRT. DNA double strand breaks and whole genome analysis were performed. Using different bioinformatics analysis, we showed that the main response after low dose exposure is the induction of immune-related response, which is composed of DNA damage and inflammatory responses, growth factor signalling and positive regulation of cell cycle. Immune responses and growth factors were shown to be related to the positive regulation of cell cycle progression; this indicates that cell cycle arrest was not activated; nevertheless, DNA damage signalling was not completed. Using quantitative RT-PCR, various genes belonging to different biological pathways were confirmed. The results of this study are presented in Chapter 7.
Monocytes are central players in the induction of immune response via the secretion of immune-stimulatory cytokines and the activation of immune-related pathways, mainly Toll-like receptors (TLRs), NF-κB and mitogen activated protein kinases (MAPKs). In an attempt to confirm the immune-stimulatory nature of the responses combined from both the in vitro and in vivo studies, we investigated the activation of immune pathways in monocytes isolated from human donors. Our results showed that low doses positively regulate the TLR, NF-κB and MAPKs signalling; however high doses did show less involvement of these pathways, which might suggest immune-suppressive responses. The results of this study are presented in Chapter 8.
The third part of this PhD thesis comprises the general discussion. It is composed of two different chapters that intend to explain all the results combined from the different studies with conclusions and perspectives.
Taking into account the results presented in Chapters 6, 7, and 8, we provide in Chapter 9 a thorough explanation about the differences between responses after low and high dose exposures. These are divided into immune responses, growth factor signalling, and damage responses. Furthermore, we discuss the involvement of the different pathways, namely MAPKs, NF-κB and growth factors in cancer development and inflammation. On the other hand, we discuss the role of NF-κB, MAPKs and growth factors as demonstrated bystander effects players. Finally, we give an overview about the different bioinformatics approaches that could be useful for whole genome analysis of low dose responses used in our studies as well as those described in the literature.
In addition to that, in Chapter 10, we conclude that low and high doses of ionizing radiation are characterized by different responses, thus extrapolation is not an accurate approach. Furthermore, we suggest that radiation protection measures and dose reducing techniques should be applied in the medical field. On the other hand, and in an attempt to develop our understanding to low dose responses, we recommend performing mechanistic studies and molecular epidemiology for clear-cut answers concerning low dose health effects. However, we discuss the many challenges that hinder the fast progression of low dose research, mainly because there is a difficulty in choosing a model for biological studies
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